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WO2015191780A2 - Interactome arn-facteur de liaison à la protéine ccctc (ctcf) - Google Patents

Interactome arn-facteur de liaison à la protéine ccctc (ctcf) Download PDF

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WO2015191780A2
WO2015191780A2 PCT/US2015/035204 US2015035204W WO2015191780A2 WO 2015191780 A2 WO2015191780 A2 WO 2015191780A2 US 2015035204 W US2015035204 W US 2015035204W WO 2015191780 A2 WO2015191780 A2 WO 2015191780A2
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oligonucleotide
ctcf
rna
nucleotides
nucleotide
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Jeannie T. Lee
Johnny KUNG
Barry Kesner
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The General Hospital Corporation
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
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    • C12N2310/531—Stem-loop; Hairpin

Definitions

  • CCCTC-Binding Factor CCCTC-Binding Factor (CTCF) RNA Interactome CLAIM OF PRIORITY
  • This invention relates to methods and compositions for selectively reactivating or repressing certain genes, e.g., genes regulated by zinc-finger protein CCCTC-binding factor (CTCF).
  • CCCTC-binding factor CCCTC-binding factor
  • Xi inactive X chromosome
  • Xi genes associated with X-linked diseases, e.g., Rett Syndrome, Factor VIII or IX deficiency, Fragile X Syndrome, Duchenne muscular dystrophy, and PNH, in heterozygous females carrying a mutated allele, in addition to a functional wildtype or hypomorphic allele.
  • CTCF The zinc-finger protein CCCTC-binding factor
  • CTCF is a ubiquitous gene regulator that is frequently mutated or aberrantly expressed in cancer and other human diseases (Lobanenkov et al., 1990; Ohlsson et al., 2001; Kim et al., 2007; Ohlsson et al., 2010; Chen et al., 2012; Nakahashi et al., 2013).
  • CTCF binds throughout the genome via combinatorial subsets of its 11 zinc fingers, serving as chromatin insulator, activator, or repressor depending on the epigenetic context (Filippova, 2008;Ong and Corces, 2014).
  • CTCF cardiovascular disease
  • CTCF intra-chromosomal regulation by CTCF
  • ICR imprint control region
  • CTCF may also aid inter-chromosomal associations between maternal H19-Igf2 ICR (Chr. 7) and paternal Wsb1-Nf1 (Chr. 11)(Ling et al., 2006), though the function of this interaction is unclear.
  • CTCF X- chromosome inactivation
  • CTCF plays a number of different roles and binds a large number of sites within concentrated regions of the X-inactivation center.
  • CTCF acts both in cis and in trans.
  • CTCF- mediated inter-chromosomal interactions play a role in X-chromosome pairing, a process that has been proposed to ensure mutually exclusive choice of active versus inactive X chromosomes (Bacher et al., 2006;Xu et al., 2006;Xu et al., 2007;Donohoe et al.,
  • CTCF-binding sites have been correlated with intra- chromosomal interactions (Tsai et al., 2008;Spencer et al., 2011) and topologically associated domains (Nora et al., 2012) around the X-inactivation center.
  • CTCF binds the RS14 element between Xist and its antisense repressor, Tsix, to form a presumptive border between two ACH’s, with one ACH involving physical interactions between promoter regions of Xist and its activator, Jpx, for the inactive X, and the other ACH centering on interactions between Tsix and its enhancer, Xite, for the active X.
  • the invention provides methods for activating an inactive X- linked allele in a cell, preferably a cell of a female heterozygous subject.
  • the methods include administering to the cell an inhibitory oligonucleotide targeting a sequence within 500 nucleotides of a CTCF binding site on a CTCF-interacting RNA, i.e., complementary or identical to a region within 500 nts of a CTCF binding site, i.e., within a sequence as listed in Tables 1-2 (each of which shows a binding site sequence +500 flanking nucleotides on both sides).
  • the inactive X-linked allele is associated with an X- linked disorder, and the oligonucleotide is administered in a therapeutically effective amount.
  • the invention provides methods for activating a repressed autosomal gene in a cell.
  • the methods include administering to the cell an inhibitory oligonucleotide targeting a sequence within 500 nucleotides of a CTCF binding site on a CTCF-interacting RNA that represses the autosome or the autosomal gene, i.e.,
  • the repressed gene is associated with a disorder, and the oligonucleotide is administered in a therapeutically effective amount.
  • the invention provides methods for downregulating an X-linked escapee gene in a cell.
  • the methods include administering to the cell an inhibitory oligonucleotide targeting a sequence within 500 nucleotides of a CTCF binding site on a CTCF-interacting RNA that increases expression of the X-linked escapee gene, i.e., complementary or identical to a region within 500 nts of a CTCF binding site on the RNA, i.e., within a sequence as listed in Tables 1-2 (each of which shows a binding site sequence +500 flanking nucleotides on both sides).
  • the X-linked escapee gene is associated with a disorder, and the oligonucleotide is administered in a therapeutically effective amount.
  • the invention provides methods for repressing an autosomal gene in a cell.
  • the methods include administering to the cell an inhibitory oligonucleotide targeting a sequence within 500 nucleotides of a CTCF binding site on a CTCF-interacting RNA that increases expression of the autosomal gene, i.e., complementary or identical to a region within 500 nts of a CTCF binding site on the RNA, i.e., within a sequence as listed in Tables 1-2 (each of which shows a binding site sequence +500 flanking nucleotides on both sides).
  • the autosomal gene is associated with a disorder, and the oligonucleotide is administered in a therapeutically effective amount.
  • the invention provides methods for increasing expression of a selected gene listed in Tables 1 or 2 in a cell; the methods include contacting the cell with a nucleic acid triplex-forming oligonucleotide (TFO) that binds specifically to a CTCF localization sequence or binding site associated with the selected gene.
  • TFO nucleic acid triplex-forming oligonucleotide
  • the cell is in a living subject, e.g., a human, and the oligonucleotide is optionally administered in a therapeutically effective amount.
  • the inhibitory oligonucleotide is identical or complementary to at least 8 consecutive nucleotides of a strong or moderate binding site nucleotide sequence as set forth in Tables 1-2, or complementary to at least 8 consecutive nucleotides of a caRNA as set forth in Tables 1-2.
  • the invention provides inhibitory oligonucleotides that are complementary or identical to at least 8 consecutive nucleotides of a CTCF binding site nucleotide sequence as set forth in Tables 1-2.
  • the oligonucleotide does not comprise three or more consecutive guanosine nucleotides.
  • At least one nucleotide of the oligonucleotide comprises a 2’ O-methyl. In some embodiments, each nucleotide of the oligonucleotide comprises a 2’ O- methyl.
  • the bridged nucleotide is a LNA nucleotide, a cEt nucleotide or a ENA modified nucleotide.
  • each nucleotide of the oligonucleotide is a LNA nucleotide. In some embodiments, one or more of the nucleotides of the oligonucleotide comprise 2’-fluoro-deoxyribonucleotides.
  • one or more of the nucleotides of the oligonucleotide comprise 2’-O-methyl nucleotides. In some embodiments, one or more of the nucleotides of the oligonucleotide comprise ENA nucleotide analogues.
  • one or more of the nucleotides of the oligonucleotide comprise LNA nucleotides.
  • the nucleotides of the oligonucleotide comprise
  • the oligonucleotide is a gapmer or a mixmer.
  • the TFO comprises one or more of DNA, RNA, PNA, HNA, MNA, ANA, LNA, CAN, INA, CeNA, TNA, (2'-NH)- TNA, (3'-NH)-TNA, alpha-L-Ribo-LNA, alpha-L-Xylo-LNA, beta-D-Ribo-LNA, beta-D- Xylo-LNA, [3.2.1]-LNA, Bicyclo-DNA, 6-Amino-Bicyclo-DNA, 5-epi-Bicyclo-DNA, alpha-Bicyclo-DNA, Tricyclo-DNA, Bicyclo[4.3.0]-DNA, Bicyclo[3.2.1]-DNA,
  • the gene is shown in Tables 1 or 2 of U.S. Provisional Patent Application Serial No. 62/010,342, filed on June 10, 2014, the entire contents of which are hereby incorporated by reference.
  • the TFO includes one or more modifications described herein.
  • This application includes a sequence listing submitted on compact disc.
  • the sequence listing is identified on the compact disc as follows.
  • FIGS. 2A-E Characterization of the CTCF RNA interactome.
  • Figures 3A-E The RNA interactome and epigenomic landscape of CTCF.
  • FIGS. 4A-C Allele-specific binding of CTCF on the X-chromosome.
  • CTCF binds RNA with high affinity and specificity.
  • Comp unlabelled competitors at 40 ⁇ molar excess. *, CTCF-RNA shift.
  • Jpx is positive control
  • Gapdh is negative control.
  • Map of Xite/Tsix and EMSA probes are shown above the gels.
  • E RNA EMSA using 1.5 pmol of purified recombinant FLAG-CTCF or FLAG- GFP and 0.5 pmol of various Tsix RNA fragments (as shown in map in panel D).
  • Comp unlabelled competitors at 40 ⁇ molar excess. *, CTCF-RNA shift.
  • CTCF full-length protein FL
  • GST-CTCF fragments N, N-terminal domain (aa 1-284); Zn, zinc-finger domain (aa 284-583); C, C-terminal domain (aa 583- 727); or GST alone.
  • Comp unlabelled competitors at 40 ⁇ molar excess. *, CTCF-RNA shift.
  • A Map of Xic and pairing center, with positions for RIP qPCR primers and EMSA probes (arrowheads).
  • Tsix antagomirs (asterisks): shRNA; LNA; LNA.
  • the Tsix major promoter accounts for 90% of Tsix transcripts. Position of the Tsix TST truncation allele is shown.
  • Xite enhancer expresses an eRNA.
  • TsixKD 263 (d0), 332 (d2), 282 (d4), 246 (d6).
  • Tsix RNA Site-specific action of Tsix RNA facilitates locus-specific targeting of CTCF.
  • POL-II transcribes Tsix RNA, which remains tethered to the site of synthesis as the RNA recruits CTCF to the locus.
  • CTCF is expressed at physiological levels in the inducible FLAG- CTCF mESC line.
  • FIGS 9A-C Metagene analysis of CLIP-seq peaks in day 3 mESC.
  • FIGS. 10A-D The CTCF RNA interactome and epigenomic landscape at additional loci.
  • Figures 11A-C Distribution of inter-Xic distances in control and Tsix- knockdown mESCs.
  • FIG. 12A-B Occupancies of CTCF and OCT4 are not significantly affected in the regions outside of the pairing center
  • A ChIP-seq analysis of SMC3, CTCF, OCT4, and H3K27me3 on X chromosome.
  • B ChIP-qPCR in shScr and shTsix at indicated sites of X chromosome. Means ⁇ 1 S.D. shown. Three independent biological replicates shown.
  • CTCF might interact with RNA on a larger scale.
  • enhancer-directed chromosomal looping involves RNA-mediated interactions (Kung et al., 2013;Lai et al., 2013b)
  • the present inventors set out to determine whether RNA may bind CTCF on a global scale and aid long-range chromatin interactions in some contexts.
  • CLIP-seq analysis was used to define an RNA interactome for CTCF in mESC and, in parallel, ChIP-seq was performed to investigate the epigenomic landscape relative to interacting transcripts.
  • One novel function of CTCF-RNA interactions described herein is the in cis locus-specific targeting of CTCF to chromatin.
  • Tsix remains tethered to the site of transcription, it serves as an allele-specific tether for CTCF and ensures locus-specific recruitment of an otherwise ubiquitous factor (Fig. 7F).
  • Tsix and Xite RNAs promote long-range chromosomal interaction.
  • imprinted noncoding RNAs such as H19, Nespas, and Gtl2 are cis-acting (Lee and Bartolomei, 2013) and interact directly with CTCF (Fig. 10A-D)
  • CTCF may be recruited to nearby imprinting control regions via a similar RNA-mediated mechanism.
  • CTCF-RNA interactions elsewhere may similarly mediate long-range interactions to form inter- and intra-chromosomal structures– structures that are now increasingly associated with CTCF binding activities (Handoko et al., 2011;Dixon et al., 2012;Sanyal et al., 2012;Shen et al., 2012;DeMare et al., 2013;Phillips-Cremins et al., 2013).
  • the CTCF-interacting RNAs may operate both in cis and in trans.
  • Tsix and Xite (Fig. 7) are cis-acting and are required to target CTCF to the pairing center of the X-inactivation center to enable long-range interactions between two X- chromosomes (inter-chromosomal pairing) and the initiation of Xist RNA expression from the future inactive X (Fig. 6DE; 7AB).
  • CTCF-associated RNAs such as Jpx (Sun et al., 2013) and SRA1 (Yao et al., 2010) are trans-acting.
  • CTCF may also function as either activator or repression of gene expression.
  • CTCF-mediated repression at the X- inactivation center, CTCF is recruited by Tsix and Xite RNA, and binding to Tsix/Xite operates as a transcriptional repressor of Xist (Fig. 6-7).
  • CTCF is believed to be acting by forming functional chromosomal domains (e.g., intra-chromosomal loops to insulate one gene while repressing or activating a neighboring gene).
  • functional chromosomal domains e.g., intra-chromosomal loops to insulate one gene while repressing or activating a neighboring gene.
  • CTCF binding sites within an interacting transcript against which antisense oligonucleotides could be designed to block CTCF-RNA
  • A“binding site” is defined as a region on the interacting RNA that makes direct contact with CTCF protein. These binding sites were identified as statistically significant“peaks” in the CLIP-seq data. Listed in Tables 1-2 are the coordinates for the genomic equivalents of the sequences of the peaks of binding PLUS 500 nucleotides of flanking sequence; the sequences are provided in the sequence listing filed herewith.
  • the additional 500 nucleotides are included because designing inhibitory nucleic acids against flanking sequences can be efficacious in targeting RNA-protein interactions (e.g., RepC RNA interaction with YY1– see Sarma et al., 2010; Jeon and Lee, 2011 ).
  • Empirically identified CTCF binding sites in the X Chromosome are described herein (see Example 5 and Table 1). These CTCF binding sites may be functionally conserved without being highly conserved at the level of overall nucleotide identity. For example, mouse Xist shows only 76% overall nucleotide identity with human XIST using sliding 21-bp windows, or an overall sequence identity of only 60%. However, within specific functional domains, such as Repeat A of XIST, the degree of conservation can be >70% between different mammalian species. The crucial motif in Repeat A is the secondary structures formed by the repeat. A CTCF binding site interacting with CTCF may therefore be similarly low in overall conservation but still have conservation in secondary structure within specific domains of the RNA, and thereby demonstrate functional conservation with respect to recruitment of CTCF.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments is at least 90% or 100%.
  • the nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • nucleic acid “identity” is equivalent to nucleic acid“homology”.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • CTCF binding sites there are several potential uses for the CTCF binding sites described herein in the expanded CTCF transcriptome: The CTCF binding sites themselves, or antagomirs and small molecules designed against them, can be utilized to modulate expression (either up or down) of CTCF target genes. In addition, the CTCF binding sites can be used to design and/or test inhibitory nucleic acids as described herein. Methods of Treatment
  • the present disclosure provides therapeutics, useful in treating a number of conditions including but not limited to various autosomal imprinting disorders, cancer, and X-linked diseases, that are formulated by designing inhibitory nucleic acids, e.g., oligonucleotides, or TFOs that bind to the CTCF binding sites as described herein (e.g., in Tables 1-2).
  • the oligo is targeted to anywhere in the binding site sequence; in some embodiments, it is targeted to a sequence within a region that starts at nt +501 from the 5’ end of a sequence in Tables 1-2, and ends at nt -501 from the 3’ end.
  • This methodology is useful particularly in X-linked disorders, e.g., in heterozygous women who retain a wildtype copy of a gene on the Xi (See, e.g., Lyon, Acta Paediatr Suppl.
  • an inhibitory nucleic acid e.g., oligonucleotide
  • administration of an inhibitory nucleic acid targeting a strong or moderate binding site is expected to prevent CTCF recruitment to a specific X-linked gene cluster or to a specific gene on the inactive X, thereby reactivating the“good” or hypomorphic copy of the X-linked gene.
  • heterozygous females are mosaic for X-linked gene expression; some cells express genes from the maternal X and other cells express genes from the paternal X.
  • the relative ratio of these two cell populations in a given female is frequently referred to as the“X-inactivation pattern.”
  • One cell population may be at a selective growth disadvantage, resulting in clonal outgrowth of cells with one or the other parental X chromosome active; this can cause significant deviation or skewing from an expected mean X-inactivation pattern (i.e., 50:50). See, e.g., Plenge et al., Am. J. Hum. Genet. 71:168–173 (2002) and references cited therein.
  • the present methods include targeting RNAs that recruit CTCF for either gene upregulation or downregulation.
  • specific genes of interest on the inactive X could be reactivated to treat X-linked diseases, when the inactivated X chromosome bears a functional or hypomorphic copy of the gene.
  • specific autosomal genes such as Igf2 could be reactivated.
  • ASOs could be targeted to CTCF to silence H19, for example.
  • the example of Fig 6-7 shows that knocking down Tsix and Xite prevented CTCF recruitment, which in turn prevent inter-chromosomal pairing and the ability to induce Xist RNA.
  • targeting the CTCF binding sites could block the expression of disease genes that“escape” from XCI.
  • the present methods can be used to treat disorders associated with X-inactivation, which includes those listed in Table A.
  • Table A was adapted in part from Germain,“Chapter 7: General aspects of X-linked diseases” in Fabry Disease: Perspectives from 5 Years of FOS. Mehta A, Beck M, Sunder- Plassmann G, editors. (Oxford: Oxford PharmaGenesis; 2006). Treating Cancer
  • compositions comprising an inhibitory nucleic acid or TFO that binds to a CTCF binding site associated with a tumor suppressor, or cancer-suppressing gene, or imprinted gene and/or other growth-suppressing genes in any of Tables 1-2.
  • cellular proliferative and/or differentiative disorders include cancer, e.g., carcinoma, sarcoma, metastatic disorders or hematopoietic neoplastic disorders, e.g., leukemias.
  • a metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of prostate, colon, lung, breast and liver origin.
  • treating includes “prophylactic treatment” which means reducing the incidence of or preventing (or reducing risk of) a sign or symptom of a disease in a patient at risk for the disease, and “therapeutic treatment”, which means reducing signs or symptoms of a disease, reducing progression of a disease, reducing severity of a disease, in a patient diagnosed with the disease.
  • treating includes inhibiting tumor cell proliferation, increasing tumor cell death or killing, inhibiting rate of tumor cell growth or metastasis, reducing size of tumors, reducing number of tumors, reducing number of metastases, increasing 1-year or 5-year survival rate.
  • cancer As used herein, the terms“cancer”,“hyperproliferative” and“neoplastic” refer to cells having the capacity for autonomous growth, i.e., an abnormal state or condition
  • Hyperproliferative and neoplastic disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state.
  • pathologic i.e., characterizing or constituting a disease state
  • non-pathologic i.e., a deviation from normal but not associated with a disease state.
  • the term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness.“Pathologic
  • hyperproliferative cells occur in disease states characterized by malignant tumor growth.
  • non-pathologic hyperproliferative cells include proliferation of cells associated with wound repair.
  • cancer or“neoplasms” include malignancies of the various organ systems, such as affecting lung (e.g., small cell, non-small cell, squamous, adenocarcinoma), breast, thyroid, lymphoid, gastrointestinal, genito-urinary tract, kidney, bladder, liver (e.g.
  • hepatocellular cancer pancreas, ovary, cervix, endometrium, uterine, prostate, brain, as well as adenocarcinomas which include malignancies such as most colon cancers, colorectal cancer, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.
  • malignancies such as most colon cancers, colorectal cancer, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.
  • carcinoma refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas.
  • the disease is renal carcinoma or melanoma.
  • Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary.
  • carcinosarcomas e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues.
  • An“adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.
  • sarcoma is art recognized and refers to malignant tumors of mesenchymal derivation.
  • proliferative disorders include hematopoietic neoplastic disorders.
  • hematopoietic neoplastic disorders includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin, e.g., arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof.
  • the diseases arise from poorly differentiated acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia.
  • myeloid disorders include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit Rev. in
  • lymphoid malignancies include, but are not limited to acute lymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM).
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • PLL prolymphocytic leukemia
  • HLL hairy cell leukemia
  • W Waldenstrom's macroglobulinemia
  • malignant lymphomas include, but are not limited to non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed- Sternberg disease.
  • cancers that can be treated using the methods described herein are listed in the categories herein, for example, and include, but are not limited to: breast, lung, prostate, CNS (e.g., glioma), salivary gland, prostate, ovarian, and leukemias (e.g., ALL, CML, or AML). Associations of these genes with a particular cancer are known in the art, e.g., as described in Futreal et al., Nat Rev Cancer. 2004; 4:177-83; and The COSMIC (Catalogue of Somatic Mutations in Cancer) database and website, Bamford et al., Br J Cancer. 2004;91:355-8; see also Forbes et al., Curr Protoc Hum Genet.
  • CNS e.g., glioma
  • salivary gland e.g., ALL, CML, or AML
  • leukemias e.g., ALL, CML, or AML.
  • the methods described herein can be used for modulating (e.g., enhancing or decreasing) pluripotency of a stem cell and to direct stem cells down specific differentiation pathways to make endoderm, mesoderm, ectoderm, and their developmental derivatives.
  • the methods include introducing into the cell an inhibitory nucleic acid that specifically binds to, or is
  • Stem cells useful in the methods described herein include adult stem cells (e.g., adult stem cells obtained from the inner ear, bone marrow, mesenchyme, skin, fat, liver, muscle, or blood of a subject, e.g., the subject to be treated); embryonic stem cells, or stem cells obtained from a placenta or umbilical cord; progenitor cells (e.g., progenitor cells derived from the inner ear, bone marrow, mesenchyme, skin, fat, liver, muscle, or blood); and induced pluripotent stem cells (e.g., iPS cells).
  • adult stem cells e.g., adult stem cells obtained from the inner ear, bone marrow, mesenchyme, skin, fat, liver, muscle, or blood
  • embryonic stem cells, or stem cells obtained from a placenta or umbilical cord e.g., progenitor cells derived from the inner ear, bone marrow, mesenchyme, skin, fat
  • CTCF-binding RNAs can be noncoding (long noncoding RNA, lncRNA) or occasionally part of a coding mRNA; for simplicity, we will refer to them together as CTCF-associated RNAs (caRNAs) henceforth.
  • inhibitory nucleic acids targeting the CTCF binding sites are used to modulate gene expression in a cell, e.g., a cancer cell, a stem cell, or other normal cell types for gene or epigenetic therapy.
  • nucleic acids used in the methods described herein are termed “inhibitory” because they inhibit the CTCF-mediated repression or enhancement of a specified gene, by binding to a CTCF-binding sequence on the caRNA itself (e.g., an antisense oligo that is complementary to the CTCF-binding region of the caRNA) or by binding to a CTCF binding site as described herein in the genome, and (without wishing to be bound by theory) preventing binding or recruitment of CTCF to the binding site and thus disrupting CTCF-mediated silencing or enhancement in the region of the binding site.
  • a CTCF-binding sequence on the caRNA e.g., an antisense oligo that is complementary to the CTCF-binding region of the caRNA
  • a CTCF binding site as described herein in the genome
  • the cells can be in vitro, including ex vivo, or in vivo (e.g., in a subject who has cancer, e.g., a tumor).
  • the methods include introducing into the cell an inhibitory nucleic acid that is modified in some way, e.g., that differs from the endogenous caRNA or CTCF binding site by including one or more modifications to the backbone or bases as described herein for inhibitory nucleic acids.
  • modified oligos are also within the scope of the present invention.
  • the methods include introducing into the cell an inhibitory nucleic acid that specifically binds, or is complementary, to a strong or moderate binding site or a long non-coding RNA described herein.
  • a nucleic acid that binds“specifically” binds primarily to the target, i.e., to the CTCF binding site to inhibit regulatory function or binding of CTCF to the caRNA or DNA but not to other non-target RNAs.
  • the specificity of the nucleic acid interaction thus refers to its function (e.g., inhibiting the CTCF- associated repression or enhancement of gene expression) rather than its hybridization capacity.
  • Inhibitory nucleic acids may exhibit nonspecific binding to other sites in the genome or other mRNAs, without interfering with binding of other regulatory proteins and without causing degradation of the non-specifically-bound RNA. Thus this nonspecific binding does not significantly affect function of other non-target RNAs and results in no significant adverse effects.
  • compositions e.g., as described herein
  • an inhibitory nucleic acid that binds to a long non-coding RNA e.g., an inhibitory nucleic acid that binds to a CTCF binding site described herein, e.g., as described in Tables 1-2
  • Tables 1-2 examples of genes involved in X-linked diseases are shown in Table A.; examples of oncogenes, tumor suppressors, and imprinted genes are shown in Tables 1-2.
  • the methods described herein can be used for modulating expression of oncogenes and tumor suppressors in cells, e.g., cancer cells.
  • the methods include introducing into the cell an inhibitory nucleic acid that binds to a CTCF-binding region of a CTCF associated RNA or DNA as described herein, that regulates the genes, e.g., the tumor suppressors, oncogenes, and/or other growth-promoting genes in Tables 1-2.
  • the methods include introducing into the cell an inhibitory nucleic acid or small molecule that specifically binds, or is complementary, to a CTCF-associated RNA targeting a tumor suppressor as set forth in Tables 1-2, e.g., in subjects with cancer, e.g., lung
  • the methods include introducing into the cell an inhibitory nucleic acid that specifically binds, or is complementary, to a CTCF- associated RNA targeting an imprinted gene as set forth in Tables 1-2,or an X-linked gene as listed in Table A.
  • a nucleic acid that binds“specifically” binds primarily to the target lncRNA or related lncRNAs to inhibit regulatory function of the lncRNA but not of other non-target RNAs.
  • the specificity of the nucleic acid interaction thus refers to its function (e.g. inhibiting the CTCF-associated repression of gene expression) rather than its hybridization capacity.
  • Inhibitory nucleic acids may exhibit nonspecific binding to other sites in the genome or other mRNAs, without interfering with binding of other regulatory proteins and without causing degradation of the non-specifically-bound RNA. Thus this nonspecific binding does not significantly affect function of other non-target RNAs and results in no significant adverse effects.
  • treating includes “prophylactic treatment” which means reducing the incidence of or preventing (or reducing risk of) a sign or symptom of a disease in a patient at risk for the disease, and “therapeutic treatment”, which means reducing signs or symptoms of a disease, reducing progression of a disease, reducing severity of a disease, in a patient diagnosed with the disease.
  • the methods described herein include administering a composition, e.g., a sterile composition, comprising an inhibitory nucleic acid that is complementary to a CTCF binding site described herein.
  • Inhibitory nucleic acids for use in practicing the methods described herein can be an antisense or small interfering RNA, including but not limited to an shRNA or siRNA.
  • the inhibitory nucleic acid is a modified nucleic acid polymer (e.g., a locked nucleic acid (LNA) molecule).
  • Inhibitory nucleic acids have been employed as therapeutic moieties in the treatment of disease states in animals, including humans. Inhibitory nucleic acids can be useful therapeutic modalities that can be configured to be useful in treatment regimens for the treatment of cells, tissues and animals, especially humans.
  • an animal preferably a human, suspected of having cancer is treated by administering an inhibitory nucleic acid in accordance with this invention.
  • the methods comprise the step of administering to the animal in need of treatment, a therapeutically effective amount of an inhibitory nucleic acid as described herein.
  • Inhibitory nucleic acids useful in the present methods and compositions include antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, siRNA compounds, single- or double-stranded RNA interference (RNAi) compounds such as siRNA compounds, molecules comprising modified bases, locked nucleic acid molecules (LNA molecules), antagomirs, peptide nucleic acid molecules (PNA molecules), and other oligomeric compounds or oligonucleotide mimetics which hybridize to at least a portion of the target nucleic acid and modulate its function.
  • RNAi RNA interference
  • the inhibitory nucleic acids include antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides comprising modified linkages, interference RNA (RNAi), short interfering RNA (siRNA); a micro, interfering RNA (miRNA); a small, temporal RNA (stRNA); or a short, hairpin RNA (shRNA); small RNA-induced gene activation (RNAa); small activating RNAs (saRNAs), or combinations thereof.
  • RNAi interference RNA
  • siRNA short interfering RNA
  • miRNA micro, interfering RNA
  • miRNA micro, interfering RNA
  • stRNA small, temporal RNA
  • shRNA short, hairpin RNA
  • small RNA-induced gene activation RNAa
  • small activating RNAs small activating RNAs (saRNAs), or combinations thereof.
  • the inhibitory nucleic acid is not an miRNA, an
  • the inhibitory nucleic acids are 10 to 50, 13 to 50, or 13 to 30 nucleotides in length.
  • this embodies oligonucleotides having antisense (complementary) portions of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length, or any range therewithin.
  • non-complementary bases may be included in such inhibitory nucleic acids; for example, an inhibitory nucleic acid 30 nucleotides in length may have a portion of 15 bases that is complementary to the targeted RNA.
  • the inhibitory nucleic acids may have a portion of 15 bases that is complementary to the targeted RNA.
  • oligonucleotides are 15 nucleotides in length.
  • the antisense or oligonucleotide compounds of the invention are 12 or 13 to 30 nucleotides in length.
  • the inhibitory nucleic acid comprises one or more modifications comprising: a modified sugar moiety, and/or a modified internucleoside linkage, and/or a modified nucleotide and/or combinations thereof. It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the modifications described herein may be incorporated in a single oligonucleotide or even at within a single nucleoside within an oligonucleotide.
  • the inhibitory nucleic acids are chimeric oligonucleotides that contain two or more chemically distinct regions, each made up of at least one nucleotide.
  • oligonucleotides typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the target) and a region that is a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids.
  • Chimeric inhibitory nucleic acids of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures comprise, but are not limited to, US patent nos.
  • the inhibitory nucleic acid comprises at least one nucleotide modified at the 2' position of the sugar, most preferably a 2'-O-alkyl, 2'-O-alkyl-O-alkyl or 2'-fluoro-modified nucleotide.
  • RNA modifications include 2'-fluoro, 2'-amino and 2' O-methyl modifications on the ribose of pyrimidines, abasic residues or an inverted base at the 3' end of the RNA.
  • modified oligonucleotides include those comprising modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain
  • oligonucleotides with phosphorothioate backbones and those with heteroatom backbones particularly CH 2 -NH- O-CH2, CH, ⁇ N(CH3) ⁇ O ⁇ CH2 (known as a methylene(methylimino) or MMI backbone], CH 2 --O--N (CH 3 )-CH 2 , CH 2 -N (CH 3 )-N (CH 3 )-CH 2 and O-N (CH 3 )- CH 2 -CH 2 backbones, wherein the native phosphodiester backbone is represented as O- P-- O- CH,); amide backbones (see De Mesmaeker et al. Ace. Chem. Res.
  • PNA peptide nucleic acid
  • Phosphorus-containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3'alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3'- amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'; see US patent nos.
  • Morpholino-based oligomeric compounds are described in Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510); Genesis, volume 30, issue 3, 2001;
  • the morpholino-based oligomeric compound is a phosphorodiamidate morpholino oligomer (PMO) (e.g., as described in Iverson, Curr. Opin. Mol. Ther., 3:235-238, 2001; and Wang et al., J. Gene Med., 12:354- 364, 2010; the disclosures of which are incorporated herein by reference in their entireties).
  • PMO phosphorodiamidate morpholino oligomer
  • Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wang et al., J. Am. Chem. Soc., 2000, 122, 8595-8602.
  • Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • These comprise those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts; see US patent nos.
  • Modified oligonucleotides are also known that include oligonucleotides that are based on or constructed from arabinonucleotide or modified arabinonucleotide residues.
  • Arabinonucleosides are stereoisomers of ribonucleosides, differing only in the configuration at the 2'-position of the sugar ring.
  • a 2'-arabino modification is 2'-F arabino.
  • the modified oligonucleotide is 2’-fluoro-D-arabinonucleic acid (FANA) (as described in, for example, Lon et al., Biochem., 41:3457-3467, 2002 and Min et al., Bioorg. Med. Chem. Lett., 12:2651 -2654, 2002; the disclosures of which are incorporated herein by reference in their entireties).
  • FANA fluoro-D-arabinonucleic acid
  • ENAs ethylene-bridged nucleic acids
  • Preferred ENAs include, but are not limited to, 2'-O,4'-C-ethylene-bridged nucleic acids.
  • LNAs examples are described in WO 2008/043753 and WO2007031091 and include compounds of the following formula.
  • the LNA used in the oligomer of the invention comprises at least one LNA unit according any of the formulas
  • Y is -O-, -S-, -NH-, or N(R H );
  • Z and Z* are independently selected among an internucleoside linkage, a terminal group or a protecting group;
  • B constitutes a natural or non-natural nucleotide base moiety, and
  • RH is selected from hydrogen and C 1-4 -alkyl.
  • the Locked Nucleic Acid (LNA) used in an oligomeric compound, such as an antisense oligonucleotide, as described herein comprises at least one nucleotide comprises a Locked Nucleic Acid (LNA) unit according any of the formulas shown in Scheme 2 of PCT/DK2006/000512 (WO2007031091).
  • LNA Locked Nucleic Acid
  • the LNA used in the oligomer of the invention comprises internucleoside linkages selected from -0-P(O)2-O-, -O-P(O,S)-O-, -0-P(S)2-O-, -S-P(O)2-O-, -S-P(O,S)-O-, -S-P(S) 2 -O-, -0-P(O) 2 -S-, -O-P(O,S)-S-, -S-P(O) 2 -S-, -O-PO(R H )-O-, O-PO(OCH 3 )-O-, -O- PO(NR H )-O-, -0-PO(OCH 2 CH 2 S-R)-O-, -O-PO(BH 3 )-O-, -O-PO(NHR H )-O-, -O-P(O) 2 - NR H -, -NR H -, -
  • thio-LNA comprises a locked nucleotide in which at least one of X or Y in the general formula above is selected from S or -CH2-S-.
  • Thio-LNA can be in both beta-D and alpha-L-configuration.
  • amino-LNA comprises a locked nucleotide in which at least one of X or Y in the general formula above is selected from -N(H)-, N(R)-, CH 2 -N(H)-, and -CH 2 -N(R)- where R is selected from hydrogen and C 1-4 -alkyl.
  • Amino-LNA can be in both beta-D and alpha- L-configuration.
  • Oxy-LNA comprises a locked nucleotide in which at least one of X or Y in the general formula above represents -O- or -CH 2 -O-. Oxy-LNA can be in both beta-D and alpha-L-configuration.
  • ena-LNA comprises a locked nucleotide in which Y in the general formula above is -CH 2 -O- (where the oxygen atom of -CH 2 -O- is attached to the 2'-position relative to the base B).
  • LNAs are described in additional detail below.
  • One or more substituted sugar moieties can also be included, e.g., one of the following at the 2' position: OH, SH, SCH 3 , F, OCN, OCH 3 OCH 3 , OCH 3 O(CH 2 )n CH 3 , O(CH 2 )n NH 2 or O(CH 2 )n CH 3 where n is from 1 to about 10; Ci to C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF3 ; OCF3; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH3; SO2 CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino;
  • a preferred modification includes 2'-methoxyethoxy [2'-0- CH 2 CH 2 OCH 3 , also known as 2'-O-(2-methoxyethyl)] (Martin et al, HeIv. Chim. Acta, 1995, 78, 486).
  • Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.
  • Inhibitory nucleic acids can also include, additionally or alternatively, nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • nucleobase often referred to in the art simply as “base”
  • “unmodified” or “natural” nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5- Me pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2'
  • oligonucleotide it is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the modifications described herein may be incorporated in a single oligonucleotide or even at within a single nucleoside within an oligonucleotide.
  • both a sugar and an internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups.
  • the base units are maintained for hybridization with an appropriate nucleic acid target compound.
  • PNA peptide nucleic acid
  • the sugar- backbone of an oligonucleotide is replaced with an amide containing backbone, for example, an aminoethylglycine backbone.
  • the nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • PNA compounds include, but are not limited to, US patent nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference . Further teaching of PNA compounds can be found in Nielsen et al, Science, 1991, 254, 1497-1500.
  • Inhibitory nucleic acids can also include one or more nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • nucleobases comprise the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases comprise other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2- thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil), 4-thiouracil, 8- halo, 8-amino, 8-thiol, 8- thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5- bromo, 5-trifluoromethyl and other
  • nucleobases comprise those disclosed in United States Patent No. 3,687,808, those disclosed in“The Concise Encyclopedia of Polymer Science And Engineering”, pages 858- 859, Kroschwitz, ed. John Wiley & Sons, 1990; those disclosed by Englisch et al.,
  • nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, comprising 2-aminopropyladenine, 5-propynyluracil and 5- propynylcytosine.
  • 5- methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2oC (Sanghvi, et al., eds,“Antisense Research and Applications,” CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications. Modified nucleobases are described in US patent nos.
  • the inhibitory nucleic acids are chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide.
  • one or more inhibitory nucleic acids, of the same or different types can be conjugated to each other; or inhibitory nucleic acids can be conjugated to targeting moieties with enhanced specificity for a cell type or tissue type.
  • moieties include, but are not limited to, lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem.
  • a thioether e.g., hexyl-S- tritylthiol (Manoharan et al, Ann. N. Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl.
  • Acids Res., 1992, 20, 533-538 an aliphatic chain, e.g., dodecandiol or undecyl residues (Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49- 54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl- rac- glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl.
  • a phospholipid e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl- rac- gly
  • conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers.
  • Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.
  • Groups that enhance the pharmacodynamic properties include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid.
  • Groups that enhance the pharmacokinetic properties include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention. Representative conjugate groups are disclosed in International Patent Application No. PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,860, which are incorporated herein by reference.
  • Conjugate moieties include, but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl- rac- glycerol or triethylammonium l,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxy cholesterol moiety.
  • lipid moieties such as a cholesterol moiety, cholic acid, a thi
  • the inhibitory nucleic acids useful in the present methods are sufficiently complementary to the target a CTCF binding site, e.g., hybridize sufficiently well and with sufficient biological functional specificity, to give the desired effect.
  • “Complementary” refers to the capacity for pairing, through base stacking and specific hydrogen bonding, between two sequences comprising naturally or non-naturally occurring (e.g., modified as described above) bases (nucleosides) or analogs thereof. For example, if a base at one position of an inhibitory nucleic acid is capable of hydrogen bonding with a base at the corresponding position of a CTCF binding site, then the bases are considered to be complementary to each other at that position. 100% complementarity is not required.
  • inhibitory nucleic acids can comprise universal bases, or inert abasic spacers that provide no positive or negative contribution to hydrogen bonding.
  • Base pairings may include both canonical Watson-Crick base pairing and non-Watson-Crick base pairing (e.g., Wobble base pairing and Hoogsteen base pairing).
  • adenosine-type bases are complementary to thymidine-type bases (T) or uracil-type bases (U), that cytosine-type bases (C) are complementary to guanosine-type bases (G), and that universal bases such as such as 3-nitropyrrole or 5-nitroindole can hybridize to and are considered complementary to any A, C, U, or T.
  • T thymidine-type bases
  • U uracil-type bases
  • C cytosine-type bases
  • G guanosine-type bases
  • universal bases such as such as 3-nitropyrrole or 5-nitroindole
  • the location on a target CTCF binding site to which an inhibitory nucleic acid hybridizes is a region to which a protein binding partner binds.
  • Routine methods can be used to design an inhibitory nucleic acid that binds to a selected strong or moderate binding site sequence with sufficient specificity.
  • the methods include using bioinformatics methods known in the art to identify regions of secondary structure, e.g., one, two, or more stem-loop structures, or pseudoknots, and selecting those regions to target with an inhibitory nucleic acid.
  • Target segments 5-500 nucleotides in length comprising a stretch of at least five (5) consecutive nucleotides within the protein binding region, or immediately adjacent thereto, are considered to be suitable for targeting as well.
  • Target segments can include sequences that comprise at least the 5 consecutive nucleotides from the 5 '-terminus of one of the protein binding regions (the remaining nucleotides being a consecutive stretch of the same RNA beginning immediately upstream of the 5'-terminus of the binding segment and continuing until the inhibitory nucleic acid contains about 5 to about 100 nucleotides).
  • preferred target segments are represented by RNA sequences that comprise at least the 5 consecutive nucleotides from the 3 '-terminus of one of the illustrative preferred target segments (the remaining nucleotides being a consecutive stretch of the same CTCF binding site beginning immediately downstream of the 3'-terminus of the target segment and continuing until the inhibitory nucleic acid contains about 5 to about 100 nucleotides).
  • hybridization means base stacking and hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases.
  • hydrogen bonding may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases.
  • adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds.
  • Complementary refers to the capacity for precise pairing between two nucleotides.
  • a base at one position of an inhibitory nucleic acid is capable of hydrogen bonding with a base at the corresponding position of a CTCF binding site, then the bases are considered to be complementary to each other at that position. 100% complementarity is not required. It is understood in the art that a complementary nucleic acid sequence need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable.
  • stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate.
  • Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide.
  • Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C.
  • Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art.
  • concentration of detergent e.g., sodium dodecyl sulfate (SDS)
  • SDS sodium dodecyl sulfate
  • Various levels of stringency are accomplished by combining these various conditions as needed.
  • hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS.
  • hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 ⁇ g/ml denatured salmon sperm DNA (ssDNA).
  • hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 ⁇ g/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
  • wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature.
  • stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.
  • Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, more preferably of at least about 42° C, and even more preferably of at least about 68° C.
  • wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS.
  • Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961 , 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.
  • the inhibitory nucleic acids useful in the methods described herein have at least 80% sequence complementarity to a target region within the target nucleic acid, e.g., 90%, 95%, or 100% sequence complementarity to the target region within a CTCF binding site.
  • a target region within the target nucleic acid
  • an antisense compound in which 18 of 20 nucleobases of the antisense oligonucleotide are complementary, and would therefore specifically hybridize, to a target region would represent 90 percent complementarity.
  • Percent complementarity of an inhibitory nucleic acid with a region of a target nucleic acid can be determined routinely using basic local alignment search tools (BLAST programs) (Altschul et al., J. Mol.
  • Target-specific effects with corresponding target-specific functional biological effects, are possible even when the inhibitory nucleic acid exhibits non-specific binding to a large number of non-target RNAs.
  • short 8 base long inhibitory nucleic acids that are fully complementary to a CTCF binding site may have multiple 100% matches to hundreds of sequences in the genome, yet may produce target-specific effects, e.g.
  • 8-base inhibitory nucleic acids have been reported to prevent exon skipping with with a high degree of specificity and reduced off-target effect. See Singh et al., RNA Biol., 2009; 6(3): 341–350. 8-base inhibitory nucleic acids have been reported to interfere with miRNA activity without significant off-target effects. See Obad et al., Nature Genetics, 2011; 43: 371–378.
  • inhibitory nucleic acids please see US2010/0317718 (antisense oligos); US2010/0249052 (double-stranded ribonucleic acid (dsRNA));
  • the inhibitory nucleic acids are antisense oligonucleotides.
  • oligonucleotides are chosen that are sufficiently complementary to the target, i.e., that hybridize sufficiently well and with sufficient biological functional specificity, to give the desired effect.
  • Modified Bases including Locked Nucleic Acids (LNAs)
  • the inhibitory nucleic acids used in the methods described herein comprise one or more modified bonds or bases.
  • Modified bases include phosphorothioate, methylphosphonate, peptide nucleic acids, or locked nucleic acids (LNAs).
  • the modified nucleotides are part of locked nucleic acid molecules, including [alpha]-L-LNAs.
  • LNAs include ribonucleic acid analogues wherein the ribose ring is“locked” by a methylene bridge between the 2’-oxgygen and the 4’-carbon– i.e., oligonucleotides containing at least one LNA monomer, that is, one 2'-O,4'-C-methylene- ⁇ -D-ribofuranosyl nucleotide.
  • LNA bases form standard Watson-Crick base pairs but the locked configuration increases the rate and stability of the basepairing reaction (Jepsen et al., Oligonucleotides, 14, 130-146 (2004)).
  • LNAs also have increased affinity to base pair with RNA as compared to DNA.
  • LNAs especially useful as probes for fluorescence in situ hybridization (FISH) and comparative genomic hybridization, as knockdown tools for miRNAs, and as antisense oligonucleotides to target mRNAs or other RNAs, e.g., CTCF binding sites as described herien.
  • the modified base/LNA molecules can be designed using any method known in the art; a number of algorithms are known, and are commercially available (e.g., on the internet, for example at exiqon.com). See, e.g., You et al., Nuc. Acids. Res. 34:e60 (2006); McTigue et al., Biochemistry 43:5388-405 (2004); and Levin et al., Nuc. Acids. Res. 34:e142 (2006).
  • “gene walk” methods similar to those used to design antisense oligos, can be used to optimize the inhibitory activity of a modified base/LNA molecule; for example, a series of oligonucleotides of 10-30 nucleotides spanning the length of a target CTCF binding site can be prepared, followed by testing for activity.
  • gaps e.g., of 5-10 nucleotides or more, can be left between the LNAs to reduce the number of oligonucleotides synthesized and tested.
  • GC content is preferably between about 30--60 %.
  • LNA sequences will bind very tightly to other LNA sequences, so it is preferable to avoid significant complementarity within an LNA molecule. Contiguous runs of three or more Gs or Cs, or more than four LNA residues, should be avoided where possible (for example, it may not be possible with very short (e.g., about 9-10 nt) oligonucleotides).
  • the LNAs are xylo-LNAs.
  • the modified base/LNA molecules can be designed to target a specific region of the CTCF binding site.
  • a specific functional region can be targeted, e.g., a region comprising a known RNA localization motif (i.e., a region complementary to the target nucleic acid on which the CTCF binding site acts), or a region comprising a known protein binding region, e.g., a CTCF binding region.
  • LNAs Locked nucleic acids
  • highly conserved regions can be targeted, e.g., regions identified by aligning sequences from disparate species such as primate (e.g., human) and rodent (e.g., mouse) and looking for regions with high degrees of identity. Percent identity can be determined routinely using basic local alignment search tools (BLAST programs) (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656), e.g., using the default parameters.
  • BLAST programs Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656
  • LNA molecules can be used as a valuable tool to manipulate and aid analysis of long nuclear ncRNAs.
  • Advantages offered by an LNA molecule-based system are the relatively low costs, easy delivery, and rapid action. While other inhibitory nucleic acids may exhibit effects after longer periods of time, LNA molecules exhibit effects that are more rapid, e.g., a comparatively early onset of activity, are fully reversible after a recovery period following the synthesis of new CTCF binding site, and occur without causing substantial or substantially complete RNA cleavage or degradation.
  • One or more of these design properties may be desired properties of the inhibitory nucleic acids of the invention.
  • LNA molecules make possible the systematic targeting of domains within much longer nuclear transcripts.
  • the methods described herein include using LNA molecules to target CTCF binding sites for a number of uses, including as a research tool to probe the function of a specific CTCF binding site, e.g., in vitro or in vivo.
  • the methods include selecting one or more desired CTCF binding sites, designing one or more LNA molecules that target the CTCF binding site, providing the designed LNA molecule, and administering the LNA molecule to a cell or animal.
  • the methods can optionally include selecting a region of the CTCF binding site and designing one or more LNA molecules that target that region of the CTCF binding site.
  • LNA molecules can be created to treat such imprinted diseases.
  • the long QT Syndrome can be caused by a K+ gated Calcium-channel encoded by Kcnq1.
  • LNA molecules can be created to downregulate Kcnq1ot1, thereby restoring expression of Kcnq1.
  • LNA molecules could inhibit caRNA binding to CTCF to reverse the imprinted defect.
  • LNA molecules or similar polymers e.g., xylo-LNAs
  • LNA molecules or similar polymers that specifically bind to, or are complementary to, CTCF binding sites can prevent recruitment of CTCF to a specific chromosomal locus, in a gene-specific fashion.
  • LNA molecules might also be administered in vivo to treat other human diseases, such as but not limited to cancer, neurological disorders, infections, inflammation, and myotonic dystrophy.
  • LNA molecules might be delivered to tumor cells to downregulate the biologic activity of a growth-promoting or oncogenic long nuclear ncRNA (e.g., Gtl2 or MALAT1 (Luo et al., Hepatology. 44(4):1012-24 (2006)), a lncRNA associated with metastasis and is frequently upregulated in cancers).
  • Repressive caRNAs downregulating tumor suppressors can also be targeted by LNA molecules to promote reexpression.
  • INK4b/ARF/INK4a tumor suppressor locus expression of the INK4b/ARF/INK4a tumor suppressor locus is controlled by Polycomb group proteins including PRC1 and PRC2 and repressed by the antisense noncoding RNA ANRIL (Yap et al., Mol Cell. 2010 Jun 11;38(5):662-74).
  • ANRIL can be targeted by LNA molecules to promote reexpression of the INK4b/ARF/INK4a tumor suppressor.
  • Some CTCF binding sites may be associated with caRNAs that are positive regulators of oncogenes. Such“activating caRNAs” have been described recently (e.g., Jpx (Tian et al., Cell. 143(3):390-403 (2010) and others ( ⁇ rom et al., Cell.
  • LNA molecules could be directed at these activating CTCF binding sites to downregulate oncogenes.
  • LNA molecules could also be delivered to inflammatory cells to downregulate regulatory caRNA that modulate the inflammatory or immune response. (e.g., LincRNA-Cox2, see Guttman et al., Nature. 458(7235):223-7. Epub 2009 Feb 1 (2009)).
  • the LNA molecules targeting CTCF binding sites described herein can be used to create animal or cell models of conditions associated with altered gene expression (e.g., as a result of altered epigenetics).
  • X chromosome changes are often seen in female reproductive cancers.
  • Some 70% of breast carcinomas lack a‘Barr body’, the cytologic hallmark of the inactive X chromosome (Xi), and instead harbor two or more active Xs (Xa).
  • Additional X’s are also a risk factor for men, as XXY men (Klinefelter Syndrome) have a 20- to 50-fold increased risk of breast cancer in a BRCA1 background.
  • the X is also known to harbor a number of oncogenes.
  • the inhibitory nucleic acid is an antagomir.
  • Antagomirs are chemically modified antisense oligonucleotides that can target a CTCF binding site.
  • an antagomir for use in the methods described herein can include a nucleotide sequence sufficiently complementary to hybridize to a CTCF binding site target sequence of about 12 to 25 nucleotides, preferably about 15 to 23 nucleotides.
  • antagomirs include a cholesterol moiety, e.g., at the 3'-end. In some embodiments, antagomirs have various modifications for RNase protection and
  • an antagomir can have one or more of complete or partial 2'-O-methylation of sugar and/or a phosphorothioate backbone. Phosphorothioate modifications provide protection against RNase or other nuclease activity and their lipophilicity contributes to enhanced tissue uptake.
  • the antagomir cam include six phosphorothioate backbone modifications; two phosphorothioates are located at the 5'-end and four at the 3'-end, but other patterns of phosphorothioate modification are also commonly employed and effective.
  • Krutzfeldt et al. describes chemically engineered oligonucleotides, termed 'antagomirs', that are reported to be efficient and specific silencers of endogenous miRNAs in mice.
  • antagomir avoids target RNA degradation due to the modified sugars present in the molecule.
  • the presence of an unbroken string of unmodified sugars supports RNAseH recruitment and enzymatic activity.
  • the design of an antagomir will include bases that contain modified sugar (e.g., LNA), at the ends or interspersed with natural ribose or deoxyribose nucleobases.
  • Antagomirs useful in the present methods can also be modified with respect to their length or otherwise the number of nucleotides making up the antagomir.
  • the antagomirs must retain specificity for their target, i.e., must not directly bind to, or directly significantly affect expression levels of, transcripts other than the intended target.
  • antagomirs may exhibit nonspecific binding that does not produce significant undesired biologic effect, e.g., the antagomirs do not affect expression levels of non-target transcripts or their association with regulatory proteins or regulatory RNAs.
  • Interfering RNA including siRNA/shRNA
  • the inhibitory nucleic acid sequence that is complementary to a CTCF binding site can be an interfering RNA, including but not limited to a small interfering RNA (“siRNA”) or a small hairpin RNA (“shRNA”).
  • interfering RNA including but not limited to a small interfering RNA (“siRNA”) or a small hairpin RNA (“shRNA”).
  • siRNA small interfering RNA
  • shRNA small hairpin RNA
  • the interfering RNA can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self- complementary (i.e., each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double stranded structure); the antisense strand comprises nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof (i.e., an undesired gene) and the sense strand comprises nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.
  • interfering RNA is assembled from a single oligonucleotide, where the self- complementary sense and antisense regions are linked by means of nucleic acid based or non-nucleic acid-based linker(s).
  • the interfering RNA can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self- complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.
  • the interfering can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating RNA interference.
  • the interfering RNA coding region encodes a self-complementary RNA molecule having a sense region, an antisense region and a loop region.
  • a self-complementary RNA molecule having a sense region, an antisense region and a loop region.
  • Such an RNA molecule when expressed desirably forms a“hairpin” structure, and is referred to herein as an“shRNA.”
  • the loop region is generally between about 2 and about 10 nucleotides in length. In some embodiments, the loop region is from about 6 to about 9 nucleotides in length.
  • the sense region and the antisense region are between about 15 and about 20 nucleotides in length.
  • the small hairpin RNA is converted into a siRNA by a cleavage event mediated by the enzyme Dicer, which is a member of the RNase III family.
  • Dicer which is a member of the RNase III family.
  • the siRNA is then capable of inhibiting the expression of a gene with which it shares homology. For details, see Brummelkamp et al., Science 296:550-553, (2002); Lee et al, Nature Biotechnol., 20, 500-505, (2002); Miyagishi and Taira, Nature Biotechnol 20:497-500, (2002); Paddison et al. Genes & Dev.
  • siRNAs The target RNA cleavage reaction guided by siRNAs is highly sequence specific.
  • siRNA containing a nucleotide sequences identical to a portion of the target nucleic acid are preferred for inhibition.
  • 100% sequence identity between the siRNA and the target gene is not required to practice the present invention.
  • the invention has the advantage of being able to tolerate sequence variations that might be expected due to genetic mutation, strain polymorphism, or evolutionary divergence.
  • siRNA sequences with insertions, deletions, and single point mutations relative to the target sequence have also been found to be effective for inhibition.
  • siRNA sequences with nucleotide analog substitutions or insertions can be effective for inhibition.
  • the siRNAs must retain specificity for their target, i.e., must not directly bind to, or directly significantly affect expression levels of, transcripts other than the intended target.
  • the inhibitory nucleic acids are ribozymes.
  • Trans-cleaving enzymatic nucleic acid molecules can also be used; they have shown promise as therapeutic agents for human disease (Usman & McSwiggen, 1995 Ann. Rep. Med. Chem. 30, 285- 294; Christoffersen and Marr, 1995 J. Med. Chem.38, 2023-2037).
  • Enzymatic nucleic acid molecules can be designed to cleave specific caRNA targets within the background of cellular RNA. Such a cleavage event renders the caRNA non-functional.
  • enzymatic nucleic acids with RNA cleaving activity act by first binding to a target RNA. Such binding occurs through the target binding portion of an enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA.
  • the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
  • RNA-cleaving ribozymes for the purpose of regulating gene expression.
  • the hammerhead ribozyme functions with a catalytic rate (kcat) of about 1 min -1 in the presence of saturating (10 MM) concentrations of Mg 2+ cofactor.
  • An artificial "RNA ligase" ribozyme has been shown to catalyze the corresponding self- modification reaction with a rate of about 100 min -1 .
  • certain modified hammerhead ribozymes that have substrate binding arms made of DNA catalyze RNA cleavage with multiple turn-over rates that approach 100 min -1 .
  • nucleic acid sequences used to practice the methods described herein can be isolated from a variety of sources, genetically engineered, amplified, and/or expressed/ generated recombinantly. If desired, nucleic acid sequences of the invention can be inserted into delivery vectors and expressed from transcription units within the vectors.
  • the recombinant vectors can be DNA plasmids or viral vectors.
  • Generation of the vector construct can be accomplished using any suitable genetic engineering techniques well known in the art, including, without limitation, the standard techniques of PCR, oligonucleotide synthesis, restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing, for example as described in Sambrook et al. Molecular Cloning: A Laboratory Manual. (1989)), Coffin et al. (Retroviruses. (1997)) and“RNA Viruses: A Practical Approach” (Alan J. Cann, Ed., Oxford University Press, (2000)).
  • inhibitory nucleic acids of the invention are synthesized chemically.
  • Nucleic acid sequences used to practice this invention can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Adams (1983) J. Am. Chem. Soc.
  • nucleic acid sequences of the invention can be stabilized against nucleolytic degradation such as by the incorporation of a modification, e.g., a nucleotide modification.
  • nucleic acid sequences of the invention includes a phosphorothioate at least the first, second, or third internucleotide linkage at the 5' or 3' end of the nucleotide sequence.
  • the nucleic acid sequence can include a 2'-modified nucleotide, e.g., a 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O- aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O- dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O--N-methylacetamido (2'-O--NMA).
  • a 2'-modified nucleotide e.g., a 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE
  • the nucleic acid sequence can include at least one 2'-O-methyl-modified nucleotide, and in some embodiments, all of the nucleotides include a 2'-O-methyl modification.
  • the nucleic acids are“locked,” i.e., comprise nucleic acid analogues in which the ribose ring is“locked” by a methylene bridge connecting the 2’-O atom and the 4’-C atom (see, e.g., Kaupinnen et al., Drug Disc. Today 2(3):287-290 (2005); Koshkin et al., J. Am. Chem. Soc.,
  • any of the modified chemistries or formats of inhibitory nucleic acids described herein can be combined with each other, and that one, two, three, four, five, or more different types of modifications can be included within the same molecule.
  • nucleic acids used to practice this invention such as, e.g., subcloning, labeling probes (e.g., random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature, see, e.g., Sambrook et al., Molecular Cloning; A
  • the inhibitory oligonucleotide comprises locked nucleic acids (LNA), ENA modified nucleotides, 2’-O-methyl nucleotides, or 2’-fluoro- deoxyribonucleotides. In some embodiments, the inhibitory oligonucleotide comprises alternating deoxyribonucleotides and 2’-fluoro-deoxyribonucleotides. In some
  • the inhibitory oligonucleotide comprises alternating deoxyribonucleotides and 2’-O-methyl nucleotides. In some embodiments, the inhibitory oligonucleotide comprises alternating deoxyribonucleotides and ENA modified nucleotides. In some embodiments, the inhibitory oligonucleotide comprises alternating deoxyribonucleotides and locked nucleic acid nucleotides. In some embodiments, the inhibitory oligonucleotide comprises alternating locked nucleic acid nucleotides and 2’-O-methyl nucleotides.
  • the oligonucleotide may comprise deoxyribonucleotides flanked by at least one bridged nucleotide (e.g., a LNA nucleotide, cEt nucleotide, ENA nucleotide) on each of the 5’ and 3’ ends of the deoxyribonucleotides.
  • the oligonucleotide may comprise
  • deoxyribonucleotides flanked by 1, 2, 3, 4, 5, 6, 7, 8 or more bridged nucleotides (e.g., LNA nucleotides, cEt nucleotides, ENA nucleotides) on each of the 5’ and 3’ ends of the deoxyribonucleotides.
  • the 5’ nucleotide of the oligonucleotide is a deoxyribonucleotide.
  • the 5’ nucleotide of the oligonucleotide is a locked nucleic acid nucleotide.
  • oligonucleotide comprise deoxyribonucleotides flanked by at least one locked nucleic acid nucleotide on each of the 5’ and 3’ ends of the deoxyribonucleotides.
  • the nucleotide at the 3’ position of the oligonucleotide has a 3’ hydroxyl group or a 3’ thiophosphate.
  • the inhibitory oligonucleotide comprises phosphorothioate internucleotide linkages. In some embodiments, the single stranded oligonucleotide comprises phosphorothioate internucleotide linkages between at least two nucleotides. In some embodiments, the single stranded oligonucleotide comprises phosphorothioate internucleotide linkages between all nucleotides.
  • oligonucleotide can have any combination of modifications as described herein.
  • the oligonucleotide may comprise a nucleotide sequence having one or more of the following modification patterns.
  • the oligonucleotide is a gapmer (contain a central stretch (gap) of DNA monomers sufficiently long to induce RNase H cleavage, flanked by blocks of LNA modified nucleotides; see, e.g., Stanton et al., Nucleic Acid Ther. 2012. 22: 344-359;
  • the oligonucleotide is a mixmer (includes alternating short stretches of LNA and DNA; Naguibneva et al., Biomed Pharmacother. 2006 Nov; 60(9):633-8; ⁇ rom et al., Gene. 2006 May 10; 372():137-41).
  • the inhibitory oligonucleotides described herein may have a sequence that does not contain guanosine nucleotide stretches (e.g., 3 or more, 4 or more, 5 or more, 6 or more consecutive guanosine nucleotides).
  • oligonucleotides having guanosine nucleotide stretches have increased non-specific binding and/or off-target effects, compared with oligonucleotides that do not have guanosine nucleotide stretches.
  • the inhibitory oligonucleotides have a sequence that has less than a threshold level of sequence identity with every sequence of nucleotides, of equivalent length, that map to a genomic position encompassing or in proximity to an off-target gene.
  • a threshold level of sequence identity may be 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity.
  • the inhibitory oligonucleotides may have a sequence that is complementary to a region that encodes an RNA that forms a secondary structure comprising at least two single stranded loops.
  • oligonucleotides that are complementary to a region that encodes an RNA that forms a secondary structure comprising one or more single stranded loops e.g., at least two single stranded loops
  • the secondary structure may comprise a double stranded stem between the at least two single stranded loops.
  • the area of complementarity between the oligonucleotide and the nucleic acid region may be at a location of the PRC2 associated region that encodes at least a portion of at least one of the loops.
  • the predicted secondary structure RNA (e.g., of the CTCF binding site) containing the nucleic acid region is determined using RNA secondary structure prediction algorithms, e.g., RNAfold, mfold. In some embodiments,
  • oligonucleotides are designed to target a region of the RNA that forms a secondary structure comprising one or more single stranded loop (e.g., at least two single stranded loops) structures which may comprise a double stranded stem between the at least two single stranded loops.
  • a single stranded loop e.g., at least two single stranded loops
  • the inhibitory oligonucleotide may have a sequence that is has greater than 30% G-C content, greater than 40% G-C content, greater than 50% G-C content, greater than 60% G- C content, greater than 70% G-C content, or greater than 80% G-C content.
  • the inhibitory oligonucleotide may have a sequence that has up to 100% G-C content, up to 95% G-C content, up to 90% G-C content, or up to 80% G-C content.
  • the inhibitory oligonucleotide may be complementary to a chromosome of a different species (e.g., a mouse, rat, rabbit, goat, monkey, etc.) at a position that encompasses or that is in proximity to that species’ homolog of the gene of interest.
  • a different species e.g., a mouse, rat, rabbit, goat, monkey, etc.
  • oligonucleotide may be complementary to a human genomic region encompassing or in proximity to the target gene and also be complementary to a mouse genomic region encompassing or in proximity to the mouse homolog of the target gene. Oligonucleotides having these characteristics may be tested in vivo or in vitro for efficacy in multiple species (e.g., human and mouse). This approach also facilitates development of clinical candidates for treating human disease by selecting a species in which an appropriate animal exists for the disease.
  • CTCF localizes/binds to genomic DNA in a sequence-specific manner; the present methods can include inhibiting this localization, in addition to or as an alternative to administering an inhibitory nucleic acid as described herein that binds a CTCF-binding RNA.
  • an inhibitory nucleic acid as described herein that binds a CTCF-binding RNA to inhibit this localization, and thus disrupt CTCF-dependent repression and increase expression of nearby genes.
  • oligonucleotides are used that bind to genomic DNA at or near (e.g., within 100, 50, or 25) nucleotides of a CTCF localization site identified in Tables 1 and 2.
  • Table 1 lists genes corresponding to the CTCF CLIP-seq peaks from human embryonic kidney cells, and Table 2 provides the Human genomic regions determined by LiftOver analysis to correspond to the CTCF CLIP-seq peaks from mus musculus mouse embryonic fibroblasts. Each table provides the SEQ ID NO: of the peak(s) (i.e., the CTCF localization site(s)) that correspond to each of the listed genes.
  • the oligonucleotides are triplex-forming oligonucleotides (TFOs).
  • TFOs are defined as triplex-forming oligonucleotides which bind as third strands to duplex DNA in a sequence specific manner.
  • Triplex-forming oligonucleotides may be comprised of any possible combination of nucleotides and modified nucleotides. Modified nucleotides may contain chemical modifications of the heterocyclic base, sugar moiety or phosphate moiety.
  • TFOs and methods of making them, are known in the art; see, e.g., Frank-Kamenetskii and Mirkin, Annual Review of Biochemistry, 64:65-95 (1995); Vasquez and Glazer, Quarterly Reviews of Biophysics, 35(01):89-107 (2002); US PGPub Nos.
  • the TFO is a single-stranded nucleic acid molecule between 5 and 100 nucleotides in length, preferably between 7 and 40 nucleotides in length, e.g., 10 to 20 or 20 to 30 nucleotides in length.
  • the base composition is homopurine or homopyrimidine, polypurine or polypyrimidine.
  • the oligonucleotides can be generated using known DNA synthesis procedures.
  • the nucleotide sequence of the oligonucleotides is selected based on a target sequence of a CTCF localization sequence as provided herein; in addition, the sequence can be determined based on physical constraints imposed by the need to achieve binding of the oligonucleotide within the major groove of the target region, and preferably have a low dissociation constant (Kd) for the oligonucleotide/target sequence.
  • Kd dissociation constant
  • the oligonucleotides should have a base composition that is conducive to triple-helix formation and can be generated based on known structural motifs for third strand binding. The most stable complexes are formed on polypurine:polypyrimidine elements, which are relatively abundant in mammalian genomes.
  • Triplex formation by TFOs can occur with the third strand oriented either parallel or anti-parallel to the purine strand of the duplex.
  • the triplets are G.G:C and A.A:T
  • the canonical triplets are C + .G:C and T.A:T.
  • the triplex structures are stabilized by two Hoogsteen hydrogen bonds between the bases in the TFO strand and the purine strand in the duplex. See U.S. Pat. No. 5,422,251 for additional information on base compositions for third strand binding oligonucleotides.
  • the TFOs can include one or more modifications, e.g., backbone modifications such as incorporation of the flexible basestacking monomers (Bulge insertions of (R)-1-O-[4-(1- pyrenylethynyl)phenylmethyl]glycerol into the middle of homopyrimidine
  • oligodeoxynucleotides (twisted intercalating nucleic acids, TINA)) as described in US PGPub No 20090216003; intercalating nucleic acid monomers as described in
  • nucleobases see, e.g., Roig and Asseline, J. Am. Chem. Soc. 2003, 125, 4416; Hildbrand et al., J. Am. Chem. Soc. 1997, 119, 5499; and Xodo et al., Nucleic Acids Res.
  • sugar moiety modifications include, but are not limited to, 2'-O-aminoetoxy, 2'-O-amonioethyl (2'-OAE), 2'-O-methoxy, 2'-O-methyl, 2-guanidoethyl (2'-OGE), 2'-O,4'-C-methylene (LNA), 2'-O-(methoxyethyl) (2'-OME) and 2'-O-(N-(methyl)acetamido) (2'-OMA).
  • 2'-O- aminoethyl sugar moiety substitutions are especially preferred; see, e.g., Carlomagno et al., J. Am. Chem.
  • Chemically-modified heterocyclic bases include, but are not limited to, inosine, 5- (1-propynyl) uracil (pU), 5-(1-propynyl) cytosine (pC), 5-methylcytosine, 8-oxo-adenine, pseudocytosine, pseudoisocytosine, 5 and 2-amino-5-(2'-deoxy-beta-D- ribofuranosyl)pyridine (2-aminopyridine), and various pyrrolo- and pyrazolopyrimidine derivatives.
  • inosine 5- (1-propynyl) uracil (pU), 5-(1-propynyl) cytosine (pC), 5-methylcytosine, 8-oxo-adenine, pseudocytosine, pseudoisocytosine, 5 and 2-amino-5-(2'-deoxy-beta-D- ribofuranosyl)pyridine (2-aminopyridine), and various pyr
  • each nucleotide monomer can be selected from the group consisting of DNA, RNA, HNA, MNA, ANA, LNA, CAN, INA, CeNA, TNA, (2'-NH)-TNA, (3'-NH)- TNA, alpha-L-Ribo-LNA, alpha-L-Xylo-LNA, beta-D-Ribo-LNA, beta-D-Xylo-LNA, [3.2.1]-LNA, Bicyclo-DNA, 6-Amino-Bicyclo-DNA, 5-epi-Bicyclo-DNA, alpha-Bicyclo- DNA, Tricyclo-DNA, Bicyclo[4.3.0]-DNA, Bicyclo[3.2.1]-DNA, Bicyclo[4.3.0]amide- DNA, beta-D-Ribopyranosyl-NA, alpha-L-Lyxopyranosyl-NA, 2'-R-RNA, 2'-OR-RNA, 2'--
  • the TFO includes a "tail” or “tail clamp” added to the Watson-Crick binding portion that binds to target strand outside the triple helix and reduces the requirement for a stretch, increasing the number of potential target sites.
  • Tail clamps added to PNAs have been described by Kaihatsu, et al., Biochemistry, 42(47):13996-4003 (2003); Bentin, et al., Biochemistry, 42(47):13987-95 (2003) Rogers, et al., Proc. Natl. Acad. Sci. USA., 99(26):16695-700 (2002)), and are known to bind to DNA more efficiently due to low dissociation constants.
  • the TFOs are modified with, or administered with, amidoanthraquinones as described in Fox et al., Proc. Natl. Acad. Sci. USA 92:7887-7891 (1995). Methods of Treatment using TFOs
  • TFOs that target CTCF binding sites associated with disease-related genes can also be used to treat subjects.
  • the DMD gene is a causal factor in Duchenne muscular dystrophy; administration of a TFO that targets a CTCF localization site associated with the XIST gene can be used to treat subjects who have Rett Syndrome.
  • One of skill in the art would be able to identify other disease-related genes from among those listed in Tables 1 and 2.
  • a TFO that targets a CTCF localization site associated with a human disease-related gene as set forth in Tables 1 or 2 (and/or Table A) can be used to treat a human having the disease to which the gene is related; in some embodiments, the TFOs are used to reactivate a normal gene in a heterozygous individual, i.e., an individual with one normal copy and one affected copy of the gene.
  • the TFO can be administered in a pharmaceutical composition or formulation as known in the art, e.g., as described herein.
  • Subjects having a genetic disease, e.g., a disease related to a gene listed in Table 1 or 2 can be identified using methods known in the art.
  • the methods described herein can include the administration of pharmaceutical
  • compositions and formulations comprising inhibitory nucleic acid sequences designed to target a CTCF binding site.
  • compositions are formulated with a pharmaceutically acceptable carrier.
  • the pharmaceutical compositions and formulations can be administered
  • compositions can be formulated in any way and can be administered in a variety of unit dosage forms depending upon the condition or disease and the degree of illness, the general medical condition of each patient, the resulting preferred method of administration and the like. Details on techniques for formulation and administration of pharmaceuticals are well described in the scientific and patent literature, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005.
  • inhibitory nucleic acids can be administered alone or as a component of a
  • composition The compounds may be formulated for administration, in any convenient way for use in human or veterinary medicine.
  • Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • Formulations of the compositions of the invention include those suitable for intradermal, inhalation, oral/ nasal, topical, parenteral, rectal, and/or intravaginal administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient (e.g., nucleic acid sequences of this invention) which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration, e.g., intradermal or inhalation.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect, e.g., an antigen specific T cell or humoral response.
  • compositions of this invention can be prepared according to any method known to the art for the manufacture of pharmaceuticals.
  • Such drugs can contain sweetening agents, flavoring agents, coloring agents and preserving agents.
  • a formulation can be admixtured with nontoxic pharmaceutically acceptable excipients which are suitable for manufacture.
  • Formulations may comprise one or more diluents, emulsifiers, preservatives, buffers, excipients, etc. and may be provided in such forms as liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled release formulations, tablets, pills, gels, on patches, in implants, etc.
  • compositions for oral administration can be formulated using
  • Such carriers enable the pharmaceuticals to be formulated in unit dosage forms as tablets, pills, powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries,
  • compositions for oral use can be formulated as a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable additional compounds, if desired, to obtain tablets or dragee cores.
  • Suitable solid excipients are carbohydrate or protein fillers include, e.g., sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose,
  • Disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
  • Push-fit capsules can contain active agents mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers.
  • the active agents can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.
  • Aqueous suspensions can contain an active agent (e.g., nucleic acid sequences of the invention) in admixture with excipients suitable for the manufacture of aqueous
  • suspensions e.g., for aqueous intradermal injections.
  • excipients include a suspending agent, such as sodium carboxymethylcellulose, methylcellulose,
  • hydroxypropylmethylcellulose sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethylene oxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensation product of ethylene oxide with a partial ester derived from fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan mono- oleate).
  • a naturally occurring phosphatide e.g., lecithin
  • the aqueous suspension can also contain one or more preservatives such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose, aspartame or saccharin.
  • preservatives such as ethyl or n-propyl p-hydroxybenzoate
  • coloring agents such as a coloring agent
  • flavoring agents such as aqueous suspension
  • sweetening agents such as sucrose, aspartame or saccharin.
  • Formulations can be adjusted for osmolarity.
  • oil-based pharmaceuticals are used for administration of nucleic acid sequences of the invention.
  • Oil-based suspensions can be formulated by suspending an active agent in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin; or a mixture of these. See e.g., U.S. Patent No.
  • the oil suspensions can contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol.
  • Sweetening agents can be added to provide a palatable oral preparation, such as glycerol, sorbitol or sucrose.
  • an antioxidant such as ascorbic acid.
  • compositions can also be in the form of oil-in-water emulsions.
  • the oily phase can be a vegetable oil or a mineral oil, described above, or a mixture of these.
  • Suitable emulsifying agents include naturally-occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan mono-oleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan mono-oleate.
  • the emulsion can also contain sweetening agents and flavoring agents, as in the formulation of syrups and elixirs. Such formulations can also contain a demulcent, a preservative, or a coloring agent.
  • these injectable oil-in-water emulsions of the invention comprise a paraffin oil, a sorbitan monooleate, an ethoxylated sorbitan monooleate and/or an ethoxylated sorbitan trioleate.
  • formulations can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at body temperatures and will therefore melt in the body to release the drug.
  • suitable non-irritating excipient which is solid at ordinary temperatures but liquid at body temperatures and will therefore melt in the body to release the drug.
  • Such materials are cocoa butter and polyethylene glycols.
  • the pharmaceutical compounds can be delivered transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.
  • the pharmaceutical compounds can also be delivered as
  • microspheres for slow release in the body can be administered via intradermal injection of drug which slowly release subcutaneously; see Rao (1995) J. Biomater Sci. Polym. Ed. 7:623-645; as biodegradable and injectable gel formulations, see, e.g., Gao (1995) Pharm. Res. 12:857-863 (1995); or, as microspheres for oral
  • the pharmaceutical compounds can be parenterally administered, such as by intravenous (IV) administration or administration into a body cavity or lumen of an organ.
  • IV intravenous
  • These formulations can comprise a solution of active agent dissolved in a pharmaceutically acceptable carrier.
  • Acceptable vehicles and solvents that can be employed are water and Ringer's solution, an isotonic sodium chloride.
  • sterile fixed oils can be employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid can likewise be used in the preparation of injectables. These solutions are sterile and generally free of undesirable matter.
  • These formulations may be sterilized by conventional, well known sterilization techniques.
  • the formulations may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
  • concentration of active agent in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, in accordance with the particular mode of administration selected and the patient's needs.
  • the formulation can be a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated using those suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation can also be a suspension in a nontoxic parenterally-acceptable diluent or solvent, such as a solution of 1,3-butanediol.
  • the administration can be by bolus or continuous infusion (e.g., substantially uninterrupted introduction into a blood vessel for a specified period of time).
  • the pharmaceutical compounds and formulations can be lyophilized.
  • Stable lyophilized formulations comprising an inhibitory nucleic acid can be made by lyophilizing a solution comprising a pharmaceutical of the invention and a bulking agent, e.g., mannitol, trehalose, raffinose, and sucrose or mixtures thereof.
  • a process for preparing a stable lyophilized formulation can include lyophilizing a solution about 2.5 mg/mL protein, about 15 mg/mL sucrose, about 19 mg/mL NaCl, and a sodium citrate buffer having a pH greater than 5.5 but less than 6.5. See, e.g., U.S. 20040028670.
  • compositions and formulations can be delivered by the use of liposomes.
  • liposomes particularly where the liposome surface carries ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the active agent into target cells in vivo. See, e.g., U.S. Patent Nos. 6,063,400; 6,007,839; Al- Muhammed (1996) J. Microencapsul. 13:293-306; Chonn (1995) Curr. Opin. Biotechnol. 6:698-708; Ostro (1989) Am. J. Hosp. Pharm. 46:1576-1587.
  • liposome means a vesicle composed of amphiphilic lipids arranged in a bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomes that are believed to interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH-sensitive or negatively-charged are believed to entrap DNA rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells.
  • Liposomes can also include "sterically stabilized" liposomes, i.e., liposomes comprising one or more specialized lipids. When incorporated into liposomes, these specialized lipids result in liposomes with enhanced circulation lifetimes relative to liposomes lacking such specialized lipids.
  • sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety.
  • PEG polyethylene glycol
  • compositions of the invention can be administered for prophylactic and/or therapeutic treatments.
  • compositions are administered to a subject who is at risk of or has a disorder described herein, in an amount sufficient to cure, alleviate or partially arrest the clinical manifestations of the disorder or its complications; this can be called a therapeutically effective amount.
  • the amount of pharmaceutical composition adequate to accomplish this is a therapeutically effective dose.
  • the dosage schedule and amounts effective for this use i.e., the dosing regimen, will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient’s physical status, age and the like. In calculating the dosage regimen for a patient, the mode of administration also is taken into consideration.
  • the dosage regimen also takes into consideration pharmacokinetics parameters well known in the art, i.e., the active agents’ rate of absorption, bioavailability, metabolism, clearance, and the like (see, e.g., Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol.58:611- 617; Groning (1996) Pharmazie 51:337-341; Fotherby (1996) Contraception 54:59-69; Johnson (1995) J. Pharm. Sci. 84:1144-1146; Rohatagi (1995) Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24:103-108; Remington: The Science and Practice of Pharmacy, 21st ed., 2005).
  • the active agents rate of absorption, bioavailability, metabolism, clearance, and the like
  • the state of the art allows the clinician to determine the dosage regimen for each individual patient, active agent and disease or condition treated.
  • Guidelines provided for similar compositions used as pharmaceuticals can be used as guidance to determine the dosage regiment, i.e., dose schedule and dosage levels, administered practicing the methods of the invention are correct and appropriate.
  • Single or multiple administrations of formulations can be given depending on for example: the dosage and frequency as required and tolerated by the patient, the degree and amount of therapeutic effect generated after each administration (e.g., effect on tumor size or growth), and the like.
  • the formulations should provide a sufficient quantity of active agent to effectively treat, prevent or ameliorate conditions, diseases or symptoms.
  • pharmaceutical formulations for oral administration are in a daily amount of between about 1 to 100 or more mg per kilogram of body weight per day.
  • Lower dosages can be used, in contrast to administration orally, into the blood stream, into a body cavity or into a lumen of an organ.
  • Substantially higher dosages can be used in topical or oral administration or administering by powders, spray or inhalation.
  • the methods described herein can include co-administration with other drugs or pharmaceuticals, e.g., compositions for providing cholesterol homeostasis.
  • the inhibitory nucleic acids can be co-administered with drugs for treating or reducing risk of a disorder described herein.
  • ⁇ UV cell pellets were resuspended in 1 -2 mL Buffer A (10 mM HEPES pH 7.9, 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM PMSF) and incubated on ice for 30 min with frequent vortexing. Nuclei were pelleted at 2500 ⁇ g for 15 min, washed in PBS, resuspended in 500 mL Buffer C (20 mM HEPES pH 7.5, 420 mM NaCl, 15% glycerol, 1.5 mM MgCl 2 , 0.5 mM PMSF, protease and RNase inhibitors) and incubated at 4qC for 30 min with rotation.
  • Buffer A 10 mM HEPES pH 7.9, 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM PMSF
  • Nuclear lysates were diluted with one volume of 20 mM HEPES pH 7.5 and treated with 40 U TURBO DNase at 37qC for 30 min to liberate chromatin-associated CTCF-RNA complexes. After quenching the DNase with 10 mM EDTA, 5% was removed and saved for RNA-seq, while the remainder was added with sarkosyl to 0.5% and the RNA was fragmented by sonication with Diagenode Bioruptor XL twice for 20 min each (with 30 s on, 30 s off cycles). Cell debris was pelleted at 16,000 ⁇ g for 10 min, the lysate was diluted again with 1 volume of 20 mM HEPES and divided into three aliquots.
  • Beads were resuspended in SDS-PAGE loading buffer at heated for 5 min at 70qC, run on 8% Bis-Tris SDS-PAGE in MOPS buffer (50 mM MOPS, 50 mM Tris, 0.1% SDS, 1 mM EDTA) at 120 V, transferred to nitrocellulose membrane, and exposed to film for autoradiography or used for immunoblot with 1:3000 ⁇ FLAG antibodies (Sigma-Aldrich F1804).
  • RNA size and quality was verified using RNA 6000 Pico chips on the Agilent Bioanalyzer.
  • CLIP-seq library was constructed from CLIP RNA using the NEBNext Small RNA Library Prep set (New England Biolabs E7330), size-selected and cleaned up of
  • primer/adaptor-dimers using Agencourt AMPure XP beads (Beckman Coulter A63880), verified with DNA High Sensitivity chips on the Agilent Bioanalyzer, quantitated using KAPA Biosystems library quantification kit (KK4844), and sequenced with the Illumina HiSeq 2000 system with 50 cycles paired end reads.
  • Second strand cDNA synthesis was performed with NEBNext mRNA Second Strand Synthesis Module (E6111) in the presence of 1.25 mM dUTP to preserve directionality. Double- stranded cDNA was then used to generate RNA-seq libraries using the NEBNext ChIP-Seq Library Prep Master Mix Set (E6240), and quality-checked and sequenced similarly as the CLIP-seq libraries.
  • ChIP was performed essentially as described (Jeon and Lee, 2011 ) using 1.5 ⁇ 10 7 TST mouse ES cells (Ogawa and Lee, 2003) and ⁇ CTCF antibodies (Cell Signaling 2899). After nuclear lysis, chromatin was sonicated in Covaris S2 ultrasonicator with 5% duty cycle, intensity 6, 200 bursts per 1-min cycle for 8 min, before IP. 15 ng of input or purified ChIP DNA was used for ChIP-seq library construction using the NEBNext ChIP-Seq Library Prep Master Mix Set. Libraries were quality-checked and sequenced similarly as CLIP-seq libraries.
  • Adaptor sequences were trimmed from libraries with either Trim Galore! v0.3.3 (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) (for CLIP-seq and RNA- seq; stringency 15 and allowed error rate 0.2) or Cutadapt v1.0 (Martin, 2011)((for ChIP- seq; allowed error rate 0.2).
  • Identical sequences were removed by custom programs prior to alignment.
  • M. mus (mus) / M. castaneus (cas) hybrid character of the ES cell lines reads were first aligned to custom mus/129 and cas genomes, and then mapped back to the reference mm9 genome (Pinter et al., 2012).
  • non-uniquely aligned fragments were put into a“reps” category.
  • mm9 RepeatMasker tracks from UCSC were obtained using the table browser. The first of each non-uniquely aligned fragment was then extracted and intersected with each family of repeat elements using BEDtools intersect with options -s, -c, and the percentage that mapped to each family was counted.
  • +UV and -UV libraries were scaled according to total number of fragments in each library (determined by SAMtools flagstat combining reads“with itself and mate mapped” and“singletons”).
  • peaks of CTCF enrichment were called using Piranha v1.2.0 (Uren et al., 2012) based on a zero-truncated Poisson distribution and a p-value cutoff of 0.01. Peaks were categorized into sense, antisense-only and intergenic classes.
  • the sense category was derived by strand-specifically intersecting peaks with 3000-nt enveloped gene bodies.
  • the antisense-only category was derived similarly using an antisense intersection and subtracting out sense genes, while the intergenic category was the result of the complement of intersecting peaks non-strand-specifically with gene bodies.
  • RNA transcripts from RNA-seq data, Cufflinks (v2.1.1) (Trapnell et al., 2012) was used on composite d0 and d3 alignments with upper-quartiles-norm normalization, and guided (-g) with mm9 Ensembl transcripts.
  • MTC mirror tag correlation
  • WTD window tag density
  • Cis-regulatory Element Annotation System (Shin et al., 2009) was used to determine peak enrichment in genomic regions and to obtain metagene profiles and metasite analyses (including comparison of CLIP peaks to ChIP enrichment).
  • Metagene profiles were obtained on .wig files generated from the peaks .bed files using a custom perl script.
  • the CEAS- associated program sitepro was used to generate metasite profiles.
  • Mus musculus CTCF cDNA with additional sequence encoding for a C-terminal 6 ⁇ His tag was cloned into pFLAG-2 (Sigma) using EcoRI and XhoI to generate pBD39.
  • GFP cDNA pFA6a-GFP(S65T)-kanMX6
  • pBD40 Recombinant FLAG-CTCF-6 ⁇ His and FLAG-GFP- 6 ⁇ His proteins were purified from Rosetta-Gami B cells (Novagen).
  • pBD39 and pBD40 transformed cells were induced with 0.2 mM IPTG at 18qC (CTCF) or 1 mM IPTG (GFP) at 30qC.
  • FLAG-GFP-6 ⁇ His expressing cells were lysed at 4qC with 50 mM sodium phosphate pH 8, 300 mM NaCl, 20 mM imidazole, 0.5% Triton X-100, and protease inhibitors. Following sonication, insoluble material was separated by centrifugation. FLAG- GFP-6 ⁇ His was purified from the supernatant using Ni-NTA resin (Qiagen).
  • FLAG-CTCF- 6 ⁇ His expressing cells were lysed at 4qC with 50 mM sodium phosphate pH 8, 300 mM NaCl, 20 mM imidazole, 0.5% Sarkosyl, and protease inhibitors. Triton X-100 was then added to 2% final (v/v). Debris was removed by centrifugation. FLAG-CTCF-6 ⁇ His was purified from the supernatant using Ni-NTA resin. Both proteins were eluted from Ni-NTA resin with 50 mM sodium phosphate pH 8, 300 mM NaCl, and 250 mM imidazole.
  • Eluates were dialyzed against 10 mM Tris-HCl pH 7.5, 2.5 mM MgCl 2 , 50 mM KCl, 0.1 mM ZnSO 4 , 1 mM DTT, 0.1% Tween-20, and 10% glycerol.
  • RNA-buffer mixture 15 ⁇ g of total RNA from day 3 female ES cells, treated with TURBO DNase and renatured by heating for 10 min 60qC followed by slow cooling to 4qC, was then incubated with the protein-beads complexes at room temperature for 1 hr in 400 ⁇ L of Binding Buffer (1 ⁇ PBS, 2 mM MgCl 2 , 0.2 mM ZnCl 2 , 0.2% NP40, 1 mM DTT, 100 U/ml RNase Inhibitor, 0.1 mg/ml yeast tRNA [Invitrogen], 0.5 mg/mL bovine serum albumin), with 10% of RNA-buffer mixture saved as input.
  • Binding Buffer 1 ⁇ PBS, 2 mM MgCl 2 , 0.2 mM ZnCl 2 , 0.2% NP40, 1 mM DTT, 100 U/ml RNase Inhibitor, 0.1 mg/ml yeast tRNA [Invitrog
  • UV-RIP was performed as previously reported (Jeon and Lee, 2011). 1 ⁇ 10 7 day 3 female ES cells were trypsinized and resuspended in PBS. Cells for +UV experiments were crosslinked with 256 nm UV in a 15-cm dish at 250 mJ/cm 2 using the Stratalinker 1800 (Stratagene).
  • UV-RIP Nuclear Isolation Buffer (10mM HEPES pH 7.5, 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM DTT, protease inhibitors) for 30 min at 4°C, after which NP40 was added to a final concentration of 0.1% and incubated for an additional 10 min at 4°C.
  • Nuclei were pelleted and lysed in 1 mL Lysis Buffer (1 ⁇ PBS, 1% NP40, 0.5% sodium deoxycholate, protease and RNase inhibitors) for 25 min at 4°C, then for 15 min at 37°C with 30 U TURBO DNase (Ambion) added.
  • Lysates were spun down at 13,000 rpm at 4°C for 10 min, and 5 ⁇ g of either ⁇ CTCF or IgG was added to the cleared lysate and incubated at 4°C overnight with rotation, saving 5% lysate as input.
  • 40 ⁇ L of Dynabeads Protein G per IP was pre-washed 3 ⁇ with Lysis Buffer, added to the lysate-antibody mixture, and incubated for 2 hr at 4°C to capture the RNA-protein- antibody complexes.
  • RNA EMSA was performed as previously described (Sun et al., 2013). Briefly, probes were in vitro transcribed with T7 RNA polymerase (Ambion) from PCR-amplified cDNA template or linearized plasmids with cloned Xist RepA or RepF sequences.
  • Transcripts were TURBO DNase treated, TRIzol purified, 5’-dephosphorylated with alkaline phosphatase (New England Biolabs), labelled with [ ⁇ - 32 P]ATP using T4 polynucleotide kinase (New England Biolabs), and cleaned with Microspin G-50 columns. Probes were denatured at 95oC for 2 min, then cooled at 70oC for 5 min, 37oC for 15 min, room temperature for 15 min, and kept in folding buffer (50 mM NaCl, 2 mM MgCl 2 ) on ice.
  • folding buffer 50 mM NaCl, 2 mM MgCl 2
  • RNA probes were incubated with recombinant proteins at room temperature for 30 min in binding buffer (total reaction volume 20 ⁇ L) containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl 2 , 0.1 mM ZnSO 4 , 10% glycerol, 0.1% Tween-20, 1 mM DTT, 1 ⁇ g poly(dI-dC), 0.1 mM polyamine, 8U RNase inhibitor (Roche), and 1 ⁇ g yeast tRNA. Samples were resolved at 4oC by TBE polyacrylamide gel electrophoresis and detected using storage phosphor screen and Typhoon scanner
  • PCR primers (unlisted primers same as those for in vitro pulldown / UV-RIP):
  • Wild-type male J1 (40XY) and female EL16.7 (40XX) ES cell lines and culture conditions have been previously described (Lee and Lu, 1999).
  • Stable knockdown cell lines were generated by linearizing a pLKO.1 -based vector containing shRNAs against Tsix, transfecting the DNA into J1 and EL16.7 cells using Lipofectamine 2000 (Invitrogen), and selecting for clones in 1 ⁇ g/mL puromycin for 7-9 days. Control cell lines containing a scrambled shRNA were also generated in parallel.
  • shRNA sequences were as follows: shTsix3 sense, 5’- GAAATAACCTCCAGAGAAATG-3’; shTsix3 antisense, 5’- CTTTATTGGAGGTCTCTTTAC-3’; shScr sense, 5’-CCTAAGGTTAAGTCGCCCTCG- 3’; shScr antisense, 5’-CGAGGGCGACTTAACCTTAGG-3’.
  • EBs embryoid bodies
  • LIF leukemia inhibitory factor
  • Antisense LNA oligonucleotides were obtained from Exiqon, Corp., and used for Tsix RNA knockdown. LNAs were delivered into female EL16.7 (40XX) ES cell line using Amaxa Biosystems Nucleofector and Mouse ES Cell Nucleofector Kit (Lonza). 2 ⁇ 10 7 trypsinized cells on d0, d3 and d6 were processed with 2uM LNA using A-24 program. Cells were collected 6h or 12h later.
  • LNA knockdown was confirmed by qRT-PCR using the following primers: TsixCf and TsixCr (see below); Tsix-F2, 5’- GTACGTTACTCGCTAGCAGTAAT-3’ and Tsix-R2, 5’- ATCCTTTGATTTTCTAATACCC-3’; b-actin F, 5’-TTCTTTGCAGCTCCTTCGTT-3’ and b-actin R, 5’-ATGGAGGGGAATACAGCCC-3’ LNA sequences are as follows: Tsix antisense LNA1 , 5’-ACTACGCAGGCATTTT-3’; Tsix antisense LNA2, 5’- GTATGGAGTCACCAGGTT-3’; Scr LNA, 5’GTGTAACACGTCTATACGCCCA-3’.
  • ChIP analyses were carried out using a modified protocol from Millipore as described (Ahn and Lee, 2010). Briefly, 1-2 ⁇ 10 7 cells were trypsinized and crosslinked with formaldehyde to a final concentration of 1% at 37qC for 10 min. Crosslinking was quenched with glycine (125 mM final), and cells were pelleted at 640 ⁇ g for 5 min and washed twice with 1 ⁇ PBS containing protease inhibitors.
  • Nuclei were isolated from fixed cells by washing once with Buffer A-NP40 (5 mM PIPES pH 8, 85 mM KCl, 0.5% NP40), incubated on ice for 10 min, then washed with Buffer A (5 mM PIPES pH 8, 85 mM KCl) and Lysis Buffer (10 mM Tris-HCl pH 8, 10 mM NaCl, 3 mM MgCl2, 0.5% NP40).
  • Buffer A-NP40 5 mM PIPES pH 8, 85 mM KCl, 0.5% NP40
  • Pelleted nuclei were resuspended in MNase buffer (10 mM Tris-HCl pH 8, 10 mM NaCl, 3 mM MgCl 2 , 1 mM CaCl 2 , 4% NP40) containing protease inhibitors and 1% SDS. Lysates were sonicated using the Diagenode Bioruptor XL (15 min total with 30 s on and 30 s off cycles). Sonicated lysates were cleared at 16,100 ⁇ g for 10 min. For each
  • Dynabeads Protein G 50 ⁇ L of Dynabeads Protein G were first incubated with 5 ⁇ g of either ⁇ CTCF, ⁇ OCT4 (Santa Cruz sc8628) or IgG for 2 hr with rotation, then with sonicated supernatants ( ⁇ 2.0 x 10 6 cells) overnight.
  • IP samples were washed 2 ⁇ each with Low Salt TSE 150 Buffer (20 mM Tris-HCl pH 8, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl), High Salt TSE 500 Buffer (20 mM Tris-HCl pH 8, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl), LiCl Buffer (10 mM Tris-HCl pH 8, 250 mM LiCl, 1% NP40, 1% deoxycholate, 1 mM EDTA), and TE Buffer (10 mM Tris-HCl pH 8, 1 mM EDTA).
  • Low Salt TSE 150 Buffer (20 mM Tris-HCl pH 8, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl
  • High Salt TSE 500 Buffer (20 mM Tris-HCl pH 8, 0.1% SDS, 1%
  • Protein-antibody complexes were eluted from the beads with freshly made Elution Buffer (50 mM Tris-HCl pH 8, 1 mM EDTA, 1% SDS, 50 mM NaHCO3) incubated at 65qC for 10 min.
  • Crosslinks were reversed by digestion with 80 ⁇ g proteinase K at 65qC for 4 hrs, and DNA was recovered by phenol/chloroform extraction and used for qPCR with the following primers (Xu et al., 2007;Donohoe et al., 2009;Navarro et al., 2010):
  • DNA FISH was performed using two X-linked probes (centromeric RP24 and pSx9-Xic) to exclude XO cells. Digital images were taken with a Nikon Eclipse 90i microscope (Nikon Instruments, Inc.) and processed using Volocity software (Improvision). X-X distances were normalized to the nuclear area as distinguished by DAPI staining of the DNA. Measurements in 3D and 2D were essentially identical because maximal z dimensions were small compared to maximal x and y (H.P. Chu and J.T. Lee, unpublished observations).
  • FISH Fluorescence in situ hybridization
  • RNA/DNA FISH was performed on ES cells as previously described (Lee and Lu, 1999) using double-stranded Xist or Chr. 2 telomeric (RP24-338B6) DNA probes labeled by nick-translation (Roche). Digital images were taken with a Nikon Eclipse 90i microscope (Nikon Instruments, Inc.) and processed using Volocity software (Improvision). Cells were counted and scored for the presence or absence of an Xist RNA cloud or checked for ploidy.
  • Example 1 The CTCF-RNA interactome
  • CTCF-RNA interactome was defined in mouse embryonic stem cell (mESC) and examine its relationship to genomic CTCF-binding sites.
  • mESC mouse embryonic stem cell
  • CLIP-seq deep sequencing
  • Fig. 9A Approximately a quarter of reads mapped more than once to the genome, of which 5-10% mapped to each of four well- characterized classes of repetitive elements, including LINEs, SINEs, LTRs, and simple repeats (Fig. 9A). Focusing on uniquely-aligned reads, the Piranha peak-caller software identified 100,000-200,000 statistically significant peaks (p ⁇ 0.01, henceforth designated “peaks”) when compared to the–UV CLIP controls. The CLIP peaks represented putative binding domains for the CTCF-RNA interactions. Using the Cis-regulatory Element Annotation System (CEAS), we determined the relative representation of CTCF CLIP peaks within genes and in intergenic space, excluding repetitive sequences.
  • CEAS Cis-regulatory Element Annotation System
  • RNA produced from ⁇ 15,000 annotated genes are targets of CTCF.
  • This large interactome is consistent with a recent analysis performed in a human osteosarcoma cell line using a different technique (Saldana-Meyer et al., 2014).
  • CTCF-interacting RNAs tend to reside within or near genes; in comparison to the reference genome, they appeared to localize preferentially to introns, exons, and 3’ untranslated regions (UTR)(Fig. 1D).
  • UTR untranslated regions
  • Antisense RNAs accounted for 2.0-2.5% of peaks, corresponding to 2,000-3,000 loci. The remaining peaks were“intergenic,” located outside of annotated genes (Table 3).
  • CTCF-binding RNAs were not enriched in any noteworthy gene ontology (GO) terms, consistent with CTCF being a global transcriptional regulator.
  • Analysis of CTCF CLIP peaks with the Multiple Em for Motif Elicitation (MEME) software also did not reveal a consensus motif, implying that CTCF recognizes RNA through secondary and/or tertiary structures, rather than through primary sequence.
  • RNA interactome lay above the diagonal, inclusive of candidate interacting transcripts such as Tsix, Xist, Nespas, H19 and Kcnq1ot1.
  • candidate interacting transcripts such as Tsix, Xist, Nespas, H19 and Kcnq1ot1.
  • the software UCSC Liftover was used to convert the empirically determined mouse mm9 coordinates to human hg19 to produce the sequences in Table 2.
  • ChIP-seq analysis of CTCF has been reported in a number of recent publications (Xi et al., 2007;Heintzman et al., 2009;Calabrese et al., 2012).
  • allelic profiling of the X-chromosome has not been carried out in female mESC during XCI.
  • ChIP-seq was performed, with biological replicates, in d0 and d3 female mESCs (Table 4).
  • Allele-specificity was made possible by a genetically marked hybrid female ES cell line carrying a disabled Tsix allele (Tsix TST /+)(Ogawa et al., 2008), ensuring that the Xi (inactive X) will be the Mus musculus X-chromosome (mus) and Xa (active X) will be the Mus castaneus X (cas). Numerous polymorphisms between mus and cas enabled us to distinguish between CTCF ChIP signals from Xi and Xa (Pinter et al., 2012). Between 60- 120 million reads were obtained for each ChIP-seq library, PCR duplicates were removed, and ChIP signals were normalized to input.
  • CTCF ChIP peaks could be observed within promoters, exons, introns, and 5’ and 3’ UTRs.
  • metagene analysis indicated a preference for promoter regions (0-3 kb upstream of TSS) and the region immediately downstream of the TTS (0-3 kb downstream of TTS) (Fig.2D).
  • RNA binding (statistically significant CLIP-seq peaks) relative to DNA binding (statistically significant ChIP-seq peaks).
  • CTCF-DNA interactions were more likely (46% vs. 5% of peaks) to occur in intergenic space.
  • DNA binding sites were often located in close proximity to CTCF-interacting transcripts (Fig. 2E,F).
  • Metagene analysis indicated that the CTCF-binding transcripts did not generally overlap the genomic binding sites (Fig. 2E).
  • CTCF tended to bind RNA sequences within gene bodies, it bound chromatin upstream of genes, preferentially in the promoter region and in regions immediately downstream of the TTS.
  • the locus for the Sox2 pluripotency factor harbors a distinct CTCF- DNA binding site upstream of its TSS and a robust CTCF-RNA interaction domain within the 3’ UTR of the Sox2 transcript (Fig. 3A).
  • the CLIP-seq profile did not resemble the RNA-seq profile; in fact, they were opposite of each other, with CLIP-seq peaks concentrated in the 3’ UTR, and RNAseq reads hsowing highest coverage in the coding region (Fig. 3A). Positive controls further confirmed CLIP-seq specificity.
  • human SRA1 RNA was previously found to associate with a protein complex containing CTCF (Yao et al., 2010).
  • our CLIP-seq analysis demonstrated that the interaction is direct and that mouse Sra1 RNA contacts CTCF via its 3 rd exon (Fig. 3B, significant peak in exon 3 [bar]).
  • binding of CTCF to Jpx RNA was previously shown to require exons 1-3 of Jpx (Sun et al., 2013).
  • Our CLIP-seq analysis not only verified direct CTCF- Jpx interactions but also revealed that contact points occur predominantly within exon 3 (Fig. 3C).
  • CTCF-Jpx RNA interactions has been proposed to cause eviction of CTCF repressor from the Xist promoter near Repeat F (RepF)(Sun et al., 2013). Indeed, we observed CTCF-DNA interactions near RepF, but also at ⁇ 1 kb upstream of the TSS and within Repeat C (RepC) (Fig. 3D). Meanwhile, CLIP-seq showed highly significant CTCF-RNA interactions within Xist exon 1, with particularly strong interactions at RepA and RepC, where PRC2 and YY1 interact with Xist/RepA transcripts (Zhao et al., 2008;Jeon and Lee, 2011 ), as well as RepF (Fig. 3D).
  • the Xi provides a complex epigenomic landscape harboring both genes that are subject to silencing and those that escape from XCI (Carrel and Willard, 2005;Li and Carrel, 2008;Yang et al., 2010;Berletch et al., 2011;Calabrese et al., 2012;Pinter et al., 2012;Mugford et al., 2014).
  • Allelic-specific ChIP-seq analysis of female mouse trophoblast stem cells (TSC) previously revealed CTCF localization patterns in a cell type with imprinted paternal X-chromosome silencing (Calabrese et al., 2012).
  • TSCs female mouse trophoblast stem cells
  • CTCF is a high-affinity and specific RNA-binding protein
  • RNA interactome for CTCF is reminiscent of Polycomb repressive complex 2 (PRC2), for which an interactome of many thousands of transcripts has been reported by RIP-seq (Zhao et al., 2010; WO 2012/065143; WO 2012/087983).
  • PRC2 Polycomb repressive complex 2
  • K d dissociation constant
  • RNAs identified in the CLIP-seq data were specifically pulled down from total cellular RNA by the purified FLAG-CTCF, whereas the negative controls, Gtl2- as and Ppia, were not pulled down by CTCF significantly (Fig. 5A). Morever, the negative control pulldowns using FLAG-GFP protein did not result in enrichment of any RNA (Fig. 5A; bars all at ⁇ 0).
  • RNA electrophoretic mobility shift assays (EMSA) with recombinant FLAG-CTCF and control FLAG-GFP proteins (Fig. 5C), testing in vitro- transcribed RNA fragments based on binding patterns informed by CLIP-seq (Fig. 3).
  • EMSA showed specific RNA-protein shifts that were abrogated by unlabelled competitors at 40 ⁇ molar excess, while control FLAG-GFP protein did not shift any of the RNA fragments (Fig. 5D).
  • CTCF-binding DNA and RNA sequences may suggest a functional relationship between the two.
  • p53 locus
  • binding of CTCF to an overlapping antisense transcript, Wrap53 regulates expression of p53 (Saldana-Meyer et al., 2014).
  • CTCF RNA interactions may have other functions in epigenetic regulation.
  • the association between CTCF and Tsix RNA was of particular interest because previous work strongly hinted that RNA may be required for inter-chromosomal interactions between the two X-inactivation centers (Xu et al., 2007).
  • Random XCI is modeled ex vivo by mESCs undergoing cell differentiation, during which they recapitulate X-chromosome counting, choice, and spreading of silencing by Xist RNA (Starmer and Magnuson, 2009;Wutz, 2011;Disteche, 2012;Lee and Bartolomei, 2013).
  • Xist is in turn controlled by the antisense Tsix locus and by Xite, a Tsix enhancer located between the major and minor promoters of Tsix and that produces short eRNAs (enhancer-associated RNAs)(Lee, 1999;Sado et al., 2001 ;Ogawa and Lee, 2003;Stavropoulos et al., 2005).
  • Tsix and Xite not only control allelic expression of Xist RNA, but also induce X-chromosome pairing, a transient event restricted to the Xic and observed prior to Xist upregulation between days 2-4 of mESC differentiation (Bacher et al., 2006;Xu et al., 2006;Xu et al., 2007;Masui et al., 2011).
  • Genetic analyses have shown that pairing depends on a 15-kb region encompassing Tsix and Xite (Xu et al., 2006;Xu et al., 2007)(Fig. 6A).
  • X-X pairing is rapidly disrupted by POL-II inhibitors, suggesting that newly synthesized transcripts— potentially Tsix and Xite themselves— may be required for the interchromosomal interactions.
  • Tsix RNA in female mESC was knocked down using various strategies, including transiently transfected siRNAs, stably expressed shRNAs, and locked nucleic acid (LNA) gapmer oligonucleotides (which access nuclear lncRNAs more effectively (Sarma et al., 2010)).
  • LNA locked nucleic acid
  • FISH DNA fluorescence in situ hybridization
  • TsixKD Knocking down Tsix (TsixKD) at all three positions reduced the number of X-X pairs between days 2-6 of differentiation (d2-d6) when pairing normally takes place (Fig. 6C-E; 11A-C).
  • scrambled shRNA or LNA did not have any effect. Because the shRNA and LNAs yielded similar results, we henceforth show experiments using cell lines expressing the stably transfected shRNA, as these cells were more amenable to long- term differentiation assays.
  • Tsix RNA may function as a locus-specific recruiting tool to recruit CTCF and thereby direct X-X pairing.
  • chromatin In TsixKD cells, chromatin
  • CTCF binding sites and their associated caRNAs were also identified in human HEK 293 kidney cells.
  • CLIP was performed to generate a CTCF-RNA interactome as described above for mESCs. Briefly, UV-crosslinking and immunoprecipitation of RNA followed by high-throughput sequencing was used to identify Ctcf-interacting RNAs. Peaks were called from uniquely mapped reads using the software Piranha.
  • CTCF a
  • CTCF mediates methylation-sensitive enhancer-blocking activity at the
  • TopHat2 accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions.
  • a steroid receptor coactivator, SRA functions as an RNA and is present in an SRC-1 complex.
  • CTCF regulates allelic expression of Igf2 by orchestrating a promoter- polycomb repressive complex 2 intrachromosomal loop.
  • HITS-CLIP yields genome-wide insights into brain alternative RNA processing. Nature 456, 464-469
  • CTCF mediates interchromosomal colocalization between Igf2/H19 and Wsb1/Nf1. Science 312, 269-272
  • CTCF is a uniquely versatile transcription regulator linked to epigenetics and disease. Trends Genet 7, 520-527 Ong, C.-T., and Corces, V.G. (2014).
  • CTCF an architectural protein bridging genome
  • CTCF regulates the human p53 gene through direct interaction with its natural antisense transcript, Wrap53.
  • CEAS cis-regulatory element
  • CTCF mediates long-range chromatin looping and local histone modification in the beta-globin locus.
  • Jpx RNA activates Xist by evicting CTCF. Cell 153, 1537-1551
  • RNA code for the FOX2 splicing regulator revealed by mapping RNA-protein interactions in stem cells. Nat Struct Mol Biol 16, 130-137

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Abstract

La présente invention concerne des procédés et des compositions permettant de réactiver ou de réguler à la baisse de façon sélective certains gènes, par exemple des gènes régulés par le facteur de liaison (CTCF) à la protéine à doigts de zinc CCCTC sur des autosomes (par exemple des gènes marqués, des gènes suppresseurs de tumeur, des gènes du cancer) et sur le chromosome X inactif (Xi), par exemple des gènes associés à des maladies liées à l'X, comme le syndrome de Rett, une déficience en facteur VIII ou IX, le syndrome de l'X fragile, la dystrophie musculaire de Duchenne et l'hémoglobinurie paroxystique nocturne, chez des sujets féminins hétérozygotes portant un allèle muté, en plus d'un allèle hypomorphe ou de type sauvage fonctionnel.
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Family Cites Families (8)

* Cited by examiner, † Cited by third party
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US20050266422A1 (en) 2002-02-20 2005-12-01 Sirna Therapeutics, Inc. Fluoroalkoxy, nucleosides, nucleotides, and polynucleotides
US20090215872A1 (en) * 2005-07-07 2009-08-27 Lee Jeannie T Methods for Controlling Stem Cell Differentiation
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EA201492116A1 (ru) * 2012-05-16 2015-05-29 Рана Терапьютикс, Инк. Композиции и способы для модулирования экспрессии mecp2

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